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Spheroid Engineering in Microfluidic Devices
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Cite this: ACS Omega 2023, 8, 4, 3630–3649
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https://doi.org/10.1021/acsomega.2c06052
Published January 18, 2023

Copyright © 2023 The Authors. Published by American Chemical Society. This publication is licensed under

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Abstract

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Two-dimensional (2D) cell culture techniques are commonly employed to investigate biophysical and biochemical cellular responses. However, these culture methods, having monolayer cells, lack cell–cell and cell–extracellular matrix interactions, mimicking the cell microenvironment and multicellular organization. Three-dimensional (3D) cell culture methods enable equal transportation of nutrients, gas, and growth factors among cells and their microenvironment. Therefore, 3D cultures show similar cell proliferation, apoptosis, and differentiation properties to in vivo. A spheroid is defined as self-assembled 3D cell aggregates, and it closely mimics a cell microenvironment in vitro thanks to cell–cell/matrix interactions, which enables its use in several important applications in medical and clinical research. To fabricate a spheroid, conventional methods such as liquid overlay, hanging drop, and so forth are available. However, these labor-intensive methods result in low-throughput fabrication and uncontrollable spheroid sizes. On the other hand, microfluidic methods enable inexpensive and rapid fabrication of spheroids with high precision. Furthermore, fabricated spheroids can also be cultured in microfluidic devices for controllable cell perfusion, simulation of fluid shear effects, and mimicking of the microenvironment-like in vivo conditions. This review focuses on recent microfluidic spheroid fabrication techniques and also organ-on-a-chip applications of spheroids, which are used in different disease modeling and drug development studies.

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1. Introduction

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Inside the body, cells have self-assembly organization capability via intercellular signaling to constitute 3D tissues hierarchically. The extracellular matrix (ECM) formed by the cells guarantees respectable properties such as cell viability, cell functionality, cell differentiation, and mechanical properties. Cell culture studies have been performed to achieve various diagnoses and treatment systems specific to the human body by imitating many physiological events that occur in the body under laboratory conditions. (1) For this purpose, cells are grown on 2D plastic surfaces, namely, Petri dishes, well plates, or specific culture flasks, with the presence of a culture medium composed of various nutrients, ions, and salts at 37 °C. Also, the culture medium is generally supplemented with serums, antibiotics, and proteins or amino acids according to the requirement of the cell being cultured. Upon cells reaching confluency, they are collected from the cultured surface by enzymes such as trypsin and are obtained as suspended. This conventional cell culture technique has still been successfully used with slight modifications since its discovery in 1907. (2) The cell culture technique is of great importance for the biomedical community, especially in tissue engineering and regenerative medicine, because it has been successfully used in preclinical research in the area pertaining to various vaccine and drug developments, cytotoxicity evaluations, biocompatibility assessments, and testing of the therapeutic effects of various molecules. (3) The key benefits of 2D cultures are that they are quite applicable, inexpensive, and not very sensitive to the changes of the operator and their environmental conditions are easily controlled. (4,5)
Although 2D cell culture techniques have still been commonly used because of the above-mentioned advantages, several drawbacks cannot be overcome, such as deteriorated cell signaling due to loss of cell phenotype, delayed response to stimuli found in the external environment due to decreased cellular polarity, and nonhomogeneous distribution of the nutrients, metabolites, signal molecules, and various gases due to the inability to imitate the microenvironment. (6,7) Because complex cellular signaling between cells and their matrix cannot be replicated in a 2D culture, in vitro experimental data obtained from 2D cultures cannot be fully represented in in vivo conditions. (8)
A 3D cell culture, which enables cells to expand and communicate in three dimensions with the surrounding extracellular milieu, has been suggested to fulfill unmet physiological needs and culture conditions in traditional cultures. (9) Basically, an optimum 3D culture should support cell growth by simultaneously providing the requisite nutrients, moisture, and oxygen and removing the degradation products. (10) 3D cultures have great advantages compared to 2D cultures in terms of cell morphology, cell differentiation, viability, cell proliferation, stimuli response, drug metabolism, gene and protein expression, cellular functionality, and in vivo relevance. (1,11) For a 3D culture, both conventional methods and new technologies based on microfluidics can be applied. Conventional methods have limited control over the size and geometry of 3D cell spheroids. (12) However, robust, reproducible, and high-throughput 3D cell spheroid formation can be achieved using microfluidic technologies. (13) Spheroids generated using advanced 3D culture techniques have been emerging as tissue precursors used to develop a variety of on-a-chip tissue and disease models, particularly drug delivery systems, by simulation of the complex multicellular architecture, barriers to mass transport, extracellular matrix synthesis, various protein and gene expressions, and in vivo physiological conditions. (14)
Although spheroid formation and its applications have been discussed in the literature, (15−18) only a few reviews examining microfluidics systems for spheroid research have been done. (19−23) Here, conventional approaches employed in the formation of spheroids are first addressed to reveal the importance of microfluidic technology in spheroid engineering. Subsequently, existing microfluidic systems utilized in the fabrication of spheroids, as well as contemporary literature examples, are presented in detail by stressing each technique’s benefits and limitations. Moreover, not only the microfluidic techniques used in spheroid production but also the diversity of cell types used in spheroid production within microfluidic systems, the parameters to be considered in culture conditions, the use of biomaterials in spheroid production, and the issues that should be considered in the design of microfluidic systems are discussed. Finally, the organ-on-a-chip applications are critically reviewed as applications of microfluidic-based chip systems used in spheroid engineering for disease modeling and drug-screening studies.

2. Conventional 3D Cell Culture Techniques Used for Spheroid Engineering

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There are many techniques proposed for 3D cell cultures, such as liquid overlay, ultralow attachment plates, liquid marbles, hanging drop, spinner bioreactors, rotational bioreactors, magnetic levitation, and gel embedding. (24,25) All these conventional techniques basically aim to provide cellular self-organization in 3D and are illustrated in Figure 1, comparatively.

Figure 1

Figure 1. Conventional 3D cell culture techniques for spheroid formation: (A) hanging drop, (B) liquid overlay, (C) spinner bioreactors, (D) rotating bioreactors, (E) ultralow attachment plates, (F) liquid marbles, (G) magnetic levitation, and (H) gel embedding.

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In the hanging-drop method, small droplets of cell suspension prepared in a culture medium are formed on the lid of a Petri dish with the help of a micropipette, and the lid is closed on a Petri dish containing water to prevent the droplets from drying (Figure 1A). Inside the hanging droplet, the cells are subjected to gravity, and microtissues are formed with the self-assembly organization. The hanging-drop technique is relatively advantageous because it enables the acquisition of microtissues in relatively uniform sizes, it is practically applicable, and the environmental conditions can be easily controlled. However, this technique is inadequate for mass production and requires intensive labor. (26)
In the liquid overlay technique, the nonadhesive substrate-coated culture surfaces are practically used without the need for special equipment so that the cells aggregate to form microtissues instead of attaching them to the surface (Figure 1B). The main limitations of this technique are the size heterogeneity of the obtained microtissues, uncontrolled cell distribution and composition, and intensive labor requirements. (27,28) In spinner bioreactors, cells in the culture medium are exposed to constant stirring, which provides shear stress that forms a 3D structure by hindering the cells from sticking to the bioreactor walls (Figure 1C). This technique is highly suitable for working with different cells at the same time to obtain heterotypic microtissue formation. It also provides scalable output, simple fabrication, and a long culture process. (29) On the other hand, the prolonged shear forces that occurred from the spinning process may drive cells to apoptosis. (30) Also, depending on the spinning speed, rupture or disintegration may arise in formed microtissues over time. (27,31) Moreover, rotating bioreactors allow cellular self-organization to obtain microtissues (32) (Figure 1D). Also, they provide long-term controllable culture conditions and allow the co-culture of various cell types. (33) Although rotating bioreactors are very effective in the transport of nutrients, the uniform distribution of different gases, and the removal of waste via the perfusion method, formed cell aggregates have nonhomogeneous size distribution. (32,33)
One conventional approach for creating spheroids is the use of ultralow attachment plates (ULA), which is based on employing hydrophobic substrates to hinder cell adhesion and cause the cells to interact with each other to form spheroids (34−36) (Figure 1E). The main limitation of this technique is the inhomogeneous size distribution of produced spheroids, which may lead to inconsistent results in experimental studies. (37)
The liquid marble (LM) technique has been developed for spheroid formation by using the surface wettability features of the material employed in a manner similar to that of the liquid overlay approach (38) (Figure 1F). In contrast to the liquid overlay approach, the goal of LM is to cover a drop of liquid with hydrophobic powder particles to create a thin, porous, elastic hydrophobic outer shell. The coating material provides a closed spheroid formation that does not support cell adhesion and allows the cells trapped inside to freely interact with each other and to self-assemble into spheroids over time. Polytetrafluoroethylene powders are the most frequently used material in the literature as a hydrophobic coating material. (39) Spheroids from different cells were successfully fabricated using LMs. (40) However, the LM technique suffers from undesirably high evaporation and is difficult to handle, which can affect the integrity, homogeneity, and size of the spheroids. (41)
Magnetic levitation, one of the 3D cell culture techniques, is based on the principle of imitating the nongravity environment with magnetic forces (42) (Figure 1G). The magnetic levitation technique may be used to form cell aggregates through positive magnetophoresis based on the directing cells labeled with magnetic beads or negative magnetophoresis based on the concept of controlling diamagnetic cells in a paramagnetic medium. (43,44) The magnetic forces used in these systems are reported to provide uniformly sized and shaped cell aggregates. (43,45,46) In addition, these microtissues, formed as a result of directed magnetic fields, can also be easily guided for tracking or imaging studies by virtue of the same magnetic forces. (47)
The gel-embedding technique is based on the encapsulation of the cells within a hydrogel defined as cross-linked polymer molecules (Figure 1H). With a wide variety of formulations, biophysical characteristics, and biological functions, hydrogels can be engineered and can thus substitute for several features of native ECMs. (48) For the preparation of hydrogels, various types of polymers, from natural to synthetic, have been used. Although natural polymers are generally preferred because of their similarity to the ECM structure, biocompatible nature, and rich protein content, synthetic polymers have often been chosen because of their adjustable mechanical and degradable properties, easy production process, and nonimmunogenic nature. (49,50) In addition, parameters such as viscoelastic property, structural integrity, stability, and degradation behavior of the developed gels should be taken into account for the formation of spheroids. (51,52)
Ultimately, the labor-intensive and time-consuming nature of these 3D conventional culture techniques, in addition to the difficulty of changing the culture medium, the low-yield production, the difficulty of controlling the spheroid size, and cross-contamination issues, have prompted researchers to investigate microfluidic approaches for spheroid production. (53,54)

3. Spheroid Engineering in Microfluidic Systems

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Spheroid formation has been applied in microfluidic systems to examine cell characteristics and microtissue formation and is used in different applications such as tissue engineering, (55) regenerative medicine, (56) drug screening, (57) and disease modeling. (58) These systems enable the use of several processes and manipulations that have typically been hard to handle with conventional methods. For instance, laminar and turbulent flow and microdroplet formation show great advantages in the formation of spheroids. (20,22) Moreover, forming 3D tissue in a dynamic environment in a microfluidic chip offers high cell viability compared to that in static conditions. (59) Microfluidic systems are also advantageous in supplying an adequate amount of nutrients and oxygen to cells in a well-controlled environment. Furthermore, with these systems, the size and composition of spheroids can be controlled precisely in a low-cost design using only a low amount of reagents. (60) Hence, microfluidic-based tools could afford great benefits in spheroid engineering compared to conventional 3D cell culture methods. Here, we will focus on different microfluidic techniques used for spheroid generation (Figure 2 and Table 1).

Figure 2

Figure 2. Illustrations for microfluidic-based methods in spheroid formation. (A) Droplet-based methods: (1) T-junction breaks up the dispersed phase with a sheath flow; (2) in co-flowing, the dispersed phase is generated with a needle or tube; and (3) flow focusing uses two sheath flows for breaking of the dispersed phase. (B) Electrowetting generated with an electrode pattern. (C) Microwells in channels. (D) Microfluidic hanging drop with open wells in the channel. (E) Microstructure for cell trapping. (F) Acoustic manipulations. (G) Dielectrophoresis with a nonuniform electrical field. (i), (ii), and (iii) represent spheroids formation steps in all images.

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Table 1. Summary of Spheroid Fabrication Techniques Used in Microfluidic Systemsa
techniquecell type usedbiomaterial type usedobtained spheroid size/volume/areano. of fabricated spheroids/spheroid formation timeapplication areareference
dropletrat hepatoma continuous cell line (H4-II-EC3)agarose72.9 ± 18.6 μm500 spheroids in 11 h3D cell culture (61)
 murine colorectal carcinoma cell line (CT26.WT)alginateup to 0.1 mm3>1000 droplets/santicancer therapies (62)
 human cervical carcinoma cells (HeLa)Matrigel and alginate138 ± 20 μmNR/96 hanticancer therapies (63)
 human mesenchymal stem cells (hMSC)alginate/arginine-glycine-aspartic acid (-RGD)30–80 μmNR/150 mintissue engineering (64)
 diffuse large B-cell lymphoma cell line (SUDHL-10); fibroblast cell line (HS-5); peripheral blood mononuclear cells (PBMCs)alginate/PuraMatrix350 ± 25 μm250 spheroids/NRdrug screening (65)
 embryo-derived teratocarcinoma cell line (P19)alginate111 μmNR/48 htissue engineering (66)
 human breast adenocarcinoma cell line (MCF-7); human mammary fibroblast cells (HMF)alginateNR200 spheroids/min /7 daysdrug screening (67)
 primary human bone-marrow-derived mesenchymal stem cells (hBMSC)polyethylene glycol-diacrylic (PEGDA)<50 μmNR/28 days3D cell culture (68)
 human glioblastoma cells (U87MG)/mouse neural stem cells (NE-4C) 100–130 μm42,000 spheroids/1 hregenerative therapy (69)
 human glioblastoma cell line (U-251)polyethylene glycol (PEG)/RGDs118–480 μmNR/1 hdrug screening (70)
 hMSCpoly(vinyl alcohol) (PVA)90 μmNR/4 weeksdrug screening (71)
 MCF-7/human fibroblast cell line (HS-5)alginate170 μm1000 spheroids/48–72 hanticancer therapies (72)
 human embryonic kidney cells (HEK293)/human bladder cancer cell line (RT4)/human epidermoid carcinoma cells (A431)PEG–perfluoropolyether (PEG–PFPE)NR85,000 spheroids/1 h3D cell culture (73)
electrowettingMadin-Darby canine kidney epithelial cells (MDCK)Geltrex/agarose/polyacrylamide/alginate/type I collagen20 μmNR/1 day3D cell culture (74)
 HepG2/mouse embryonic fibroblast cells (NIH-3T3)type I collagenNRNRdrug screening (75)
 mouse bone-marrow-derived mesenchymal stem cells (BM-mMSC)/ HT-29 up to 400 μmNR/in 72 h3D cell culture (76)
microwellHeLa/human umbilical vein endothelial cells (HUVECs)PEG50–300 μmNR/up to 36 hanticancer therapies (77)
 human dermal fibroblasts (hDFs)cellulose nanocrystals and gelatin∼150 μm2400 spheroids/5 daydrug screening (78)
 MCF-7agarose200–600 μmup to 175 spheroids/72 hdrug screening (79)
 human lung carcinoma epithelial cell line (A549)/ human osteoblasts/patient-derived spine metastases cells (BML) up to 250 μm10 spheroids/5 daypersonalized medicine (80)
 human high-grade glioma cells (UVW)/human prostate cancer cell line (LNCaP)/patient biopsy-derived prostate cancer cells 50–150 μm240 spheroids/48 hpersonalized medicine (81)
 rat embryonic fibroblast cells (REF52)/Madin-Darby canine kidney (MDCK) cellsfibronectin/collagen40–100 μmNRtissue engineering (82)
 human colorectal adenocarcinoma cell line (HT29) up to 250 μm25 spheroids/7 daysdrug screening (83)
 human colorectal carcinoma cell line (HCT116)/human glioma cell line (U87) 150–200 μm50 spheroids/48 hdrug screening (84)
 HT-29 cells 130–250 μm20 spheroids/6 daysanticancer therapies (85)
 MCF-7/U87agaroseup to 500 μm40 spheroids/5 daysanticancer therapies (86)
 human hepatocellular carcinoma cells (HepG2-C3A)gelatin methacryloyl (GelMA)191 ± 10 μm10,000 spheroids/5 daysdrug screening (87)
 human lung adenocarcinoma cells (A549)/human lung fibroblast cells (MRC-5)type I collagenNR28 spheroids/72 hregenerative therapy (88)
 human articular cartilage cells (hACs) NRNR/14 daystissue engineering (13)
 murine ES cell (ES-D3)/human hepatocellular carcinoma cell (HepG2)/monkey kidney epithelial fibroblast (COS-7) up to 210 μm5000 spheroids/24 h3D cell culture (89)
 human glioma cell line (U87)PEGDA361.3 ± 36.2 μm24 spheroids/24–48 hdrug screening (90)
 human mesenchymal stem cells (hMSC)chitosan/polydopamineup to 500 μmNR/5 days3D cell culture (91)
 human metastatic breast adenocarcinoma cell line (MDA-MB-231)/human nontumorigenic mammary epithelial cell line (MCF-10A)type I collagen100 μm1296 spheroids/5–6 days3D cell culture (92)
 MCF-7/HCT-116 180 μm240 spheroids/24 h3D cell culture (93)
 human colon adenocarcinoma cell line (Caco-2)/normal human dermal fibroblasts (NHDF)/human alveolar basal adenocarcinoma cell line (A549)/human hepatocellular carcinoma cell line (HepG2) up to 828.7 ± 49.5 μm360 spheroids/72 htissue engineering and drug screening (94)
 human hepatoma cells (Huh-7)Geltrex160 μm120 spheroids/24 hdrug screening (95)
 A549/human fetal lung fibroblast cell line (MRC-5)type I collagen142.3 μm284 spheroids/24 htissue engineering (96)
hanging dropmouse embryonic stem cells (mESC)/human lung cancer cell line (A541)/human leukemia cell line (HL-CZ) up to 250 μm234 spheroids/24 h3D cell culture (97)
 mouse embryonic stem cells (mESCs) and human MDA-MB-231 and MCF-7 breast cancer cellsMatrigelup to 730 ± 27 μm16 spheroids/10 days3D cell culture (98)
 human epithelial carcinoma cell (A431.H9) up to 0.16 μL384 spheroids/7 daysdrug screening (99)
 mouse embryonic stem cells (mESCs; ES-D3) 80–120 μm72 spheroids/24 htissue engineering (100)
 human Wharton’s jelly-derived mesenchymal stem cells (WJ-MSC) up to 500 μm24 spheroids/7 h3D cell culture (101)
 human umbilical cord blood-derived MSCs/mouse podocyte cells up to 500 μm49 spheroids/24 htissue engineering (102)
 human glioblastoma cell lines (LN229 and PDX) 4 nL900 spheroids/1 hdrug screening (103)
 human synovial sarcoma-derived cell line (HS-SY-II)/human umbilical cord-derived mesenchymal stem cells (UC-MSCs) NRNR/24 hanticancer therapies (104)
 human hepatocellular carcinoma cell line (MHCC97-H) 522 ± 40 μm26 spheroids/3 days3D cell culture (105)
microstructuresHepG2/mouse fibroblast cells (Balb/c-3T3)PEGDANR56 spheroids/48 hdrug screening (106)
 MCF-7/HepG2 up to 1 × 10–2 mm316 spheroids/2 daysdrug screening (107)
 human glioblastoma cell line (U87-MG)gelatin-based electrospun nanofibers220 μmNR/2 daysanticancer therapies (108)
 human breast tumor cells (LCC6/Her-2)alginate250 μmNR/4 daysanticancer therapies (109)
 human breast cancer cell lines (BT49 and T47D)basement membrane extract (BME)120 μm/125 μm11 spheroids/1 daydrug screening (110)
 HT29 human colon carcinoma 200–550 μm∼10000 spheroids/10 daydrug screening (111)
 human alveolar basal adenocarcinoma cell line (A549) NR16 spheroids/72 h3D cell culture (112)
 human breast cancer stem cells (BCSCs)MatrigelNRNR/5 daystissue engineering (113)
acousticMCF-7/A549/human ovarian cancer cell line (A2780)/murine embryonic carcinoma cell line (P19) NR6000 spheroids/24 h3D cell culture (114)
 murine endothelial cell line (2H11)/ NIH 3T3/human embryonal kidney cell line (293FT) 185.2 ± 50 μmNR/9 htissue engineering (115)
dielectrophoresishuman T lymphocyte cells (Jurkat)/mouse stromal cells (AC3) at least 100 μmNR/5 min3D cell culture (116)
 human hepatoma cell line (HuH7) 50–100 μmNR/45 min3D cell culture (117)
a

NR: not reported.

3.1. Droplet-Based Methods

In biomedical studies, droplet-based microfluidic methods have been used for different applications where size-controllable monodisperse droplets can be generated and manipulated (Figure 2A). (19) To create an identical spheroid, the fabrication of uniform-sized droplets is crucial. The spheroids are formed by encapsulating cells into the droplets, and the size of spheroids can be regulated by droplet size. The geometry of the microfluidic chips controls the droplet generation, and the rate of generation can be regulated by the type of fluid inside the microchannels. (61) Encapsulating cells and forming spheroids in such microfluidic systems can be achieved by using different liquids to form single- or double-emulsion droplets. (62) Single emulsions are the most basic type of emulsion, consisting of a liquid droplet dispersed in another fluid and frequently stabilized by surfactants. Water-in-oil systems, such as “cell suspension droplets in oil”, are well-known examples of single-emulsion techniques. (63) Double emulsions are liquid dispersion systems, also known as emulsions of emulsions, in which droplets of one dispersed liquid (emulsion, microemulsion, liposome, etc.) are dispersed in another liquid (water or oil), resulting in double-layered liquid droplets. (118,119) Water-in-oil-in-water systems, such as “cell suspension droplets-in-oil-in-water” are the most commonly applied double-emulsion techniques. (64) The selection of the culture medium, physiological fluid, or hydrogel for the entrapment of cells and the determination of the oil phase is of crucial importance for generating 3D cell-laden spheroids. (19) To accomplish spheroid formation in microfluidic systems, geometric models such as a T-junction, co-flowing, and flow focusing have been widely investigated. (22,61−73)
A T-junction is the most used geometry for its simple fabrication, operability, and controllability for creating monodisperse droplets. Principally, the T-junction arrangement enables the formation of droplets by the shear stress produced by two immiscible flowing fluids at the intersection of two consecutive microchannels. (120) Here, two immiscible liquids are defined as the continuous phase and the other as the dispersed phase. The intersection of two distinct microchannels carrying the continuous and dispersed phases can be positioned between 0° and 90°. (121) The continuous phase is usually composed of oils or organic solvents that are more viscous than water and immiscible with water, whereas the dispersed phase is usually water or water-based physiological fluids. (122) T-junction methods have previously been used in forming identical droplets for both cells and drug encapsulations in microfluidic platforms for drug-screening applications. (57) In a previous study, the combination of an alginate and PuraMatrix hydrogel system was used in a droplet-integrated microfluidic chip to entrap the cancer cells, fibroblasts, and lymphocytes at the same time to achieve tumor spheroids for immunotherapy studies. (65) Lenalidomide anticancer drug was tested on the obtained tumor spheroids at a size of 350 ± 25 μm. On the basis of the findings, the microfluidic system allows not only spheroid production but also measurement of cell proliferation, cell–cell interaction, and cytotoxic effects of anticancer drugs on formed spheroids semiautomatically.
The co-flowing approach allows the formation of micrometer-sized droplets by interbedding the dispersed phase and continuous phase together. (123) The phase difference between the continuous and dispersed phases causes the continuous phase to surround the dispersed phase, resulting in the formation of droplets. (124) A microfluidic platform possessing a hillock structure based on the co-flowing technique was designed to investigate the spheroid formation in alginate microcapsules. (66) The developed system was also used for the observation of the differentiation of P19 mouse embryonic carcinoma cells. It was reported that spheroids could be obtained in as little as 2 days in massive and uniform microcapsules with an average diameter of 111 μm.
In the flow-focusing technique, droplets are generated by orthogonally positioned channels that enable the droplets to flow in the direction of the dispersed phase channel. (125) Briefly, unlike the co-flowing method, two vertical channels join the main flow channel at the same point in this technique, and droplet formation occurs at this cross-junction. (126) In a study, size-controllable alginate-based spheroids were reported to possess MCF-7 tumor cells in the core and human mammary fibroblast cells (HMF) in the shell phase. (67) The efficacies of two different anticancer drugs (Paclitaxel and Curcumin) on these produced spheroids were evaluated. The microfluidic-based fabricated spheroids exhibited higher drug resistance than the monolayer cell culture models but displayed equal resistance to those spheroids prepared using conventional methods.

3.2. Electrowetting Approaches

To construct discrete droplets, electrowetting approaches are used with microelectrode arrays (Figure 2B). (127) The formation, mixing, and transportation of droplets are simple fluidic processes in digital microfluidics (DMF) using the electrowetting (EW) phenomenon without the use of pumping systems. (128) Because of their easy automation/integration, rapid analysis, reduced sample volume, and addressability, DMF methods are advantageous when compared to conventional methods. (129) Although digital microfluidics and electrowetting have not been widely used for spheroid manufacturing in recent years, particularly in terms of mass production, a few literature examples have been considered remarkable as a proposed approach for spheroid fabrication. For instance, DMF was used to form microgel on-demand arrays for constructing and culturing mesoscale cell spheroids. (74) Madin-Darby canine kidney (MDCK) cells were seeded in microgels composed of Geltrex, type I collagen, and agarose, and these microgels were separately located in the hydrophilic site of the DMF device and were cultured for 4 days for 3D cell aggregation. In another study, the DMF system was developed to form organoids for drug-screening applications. (75) Collagen scaffolds with a co-culture of HepG2 and NIH-3T3 cells were electrodynamically injected into the presented device, and organoids were formed. Afterward, acetaminophen (APAP) in different concentrations was introduced into the DMF platform, in which organoids were observed in terms of apoptosis and necrosis. According to the results, 10 mM APAP showed an apoptotic response, and both apoptotic and necrotic responses were observed for 20 mM APAP. In another study, an automated hanging-drop method with DMF was also presented for culturing bone-marrow-based mouse mesenchymal stem cell spheroids. (76) For cell manipulation, this device contained top and bottom plates having electrodes and ground electrodes, respectively. The hanging drop formation was generated on the bottom plate with holes where cells tend to aggregate. With use of this method, spheroids with high viability and uniform size (up to 400 μm) were generated.

3.3. Microwell-Based Method

Microwell-based methods have generally been preferred because of their simple and easy-to-use process. (77) With use of microfabrication processes such as photolithography, soft lithography, and etching, it is possible to create microwell arrays from different materials such as polyethylene glycol (PEG) and polydimethylsiloxane (PDMS) (Figure 2C). (130) Because the cells do not attach to the surface in the microwells made in the appropriate dimensions, spontaneous spheroid formation occurs with sedimentation and accumulation of cells in these wells. (131) In microwell-based approaches, both static and dynamic conditions, the deterioration of the structural integrity or cell loss may occur in developed spheroids during cell seeding, culture medium replacement, or various washing steps. However, the modification of the microfluidic channel dimensions (increased width or decreased height), revision of the reservoir geometry, changing of the microwell material, and use of integrated pumps may overcome these problems. (79,132−134) Collection of the formed spheroids from the microwells is essentially required for postprocessing analysis such as biochemical, differentiation, or flow cytometry analysis. In this context, a suitable flow rate can be chosen to apply an adequate lift force for spheroid collection. (135) Numerous studies have been conducted utilizing microwell-based approaches to generate uniformly sized spheroid structures in many fields, including 3D culture, drug screening, and tissue engineering. (13,77,79,81−91,93−96) For instance, the construction of colorectal cancer cell (HT29) spheroids that have approximately 250 μm diameter at the end of the 7-day culture period was reported in a reversible microfluidic platform. (85) Top and bottom parts were held together via cubic-shaped magnets that faced each other with opposite magnetic polarity. Alternatively, another microfluidic platform has reported the formation of multicellular spheroids having 50–100 μm diameter from biopsy-derived patient cells for drug-screening studies. (81) This platform offers a self-perfusion ability that makes the platform an equipment-free device for the cultivation of formed spheroids. Furthermore, the concave microwells were used to form colon cancer (HCT116) cell spheroids having 120 μm diameter for the application of Irinotecan anticancer drug. (84) It was reported that the cell viability, the spheroid number, and the spheroid uniformity were altered depending on the concentration level of the drug. Recently, tumor spheroids were formed using a human colon cancer cell line (HT29) in microwells under a continuous flow of culture medium. (83) The developed platform allowed spheroid formations up to 250 μm diameter in size, and the cell viability was drastically decreased in spheroids throughout the 5-day culture in the presence of 5-fluorouracil anticancer drug. In another study, self-filling agarose-based microwells were demonstrated using inclined channels for analyzing drug toxicity in spheroids formed with MCF-7 breast cancer cells and also U87 brain tumor cells. (86) It was shown that the developed tumor spheroids were found more resistant to the cytotoxic effect of the doxorubicin anticancer drug than the monolayer culture of the same cells. In another study, dense dermal fibroblast spheroids were created in biomimetic cellulose-nanocrystals-doped gelatin hydrogel under physiological flow conditions and to screen for alimunium (Al) in skin care products. (78) It has been reported that 2400 spheroids with a diameter of 150 μm can be produced within 5 days in the proposed microfluidic platform. A microwell-based microfluidic biochip was also designed to generate uniform multicellular spheroids to study chemotherapeutic drugs. (80) Ten spheroids with a size of 250 μm can be formed in 5 days on this chip and formed spheroids exhibit high resistance to anticancer drugs.

3.4. Hanging Drop

The hanging-drop method is known to be one of the widely used conventional methods for 3D spheroid formation in various applications. (97,100−105) Becaue this easy-to-use method allowed the self-assembly of spheroids with the force of gravity and desired microenvironment for spheroids, the hanging-drop method has also been adapted to microfluidic systems (Figure 2D). (136) For example, a PDMS-based microfluidic hanging-drop chip was reported to provide an automated long-term and high-throughput 3D cell culture for various applications such as cell differentiation, tissue engineering, developmental biology, and drug screening. (97) Moreover, spheroids composed of Wharton’s jelly mesenchymal stromal cells (WJ-MSCs) were formed in a continuously perfused microfluidic hanging-drop platform. (101) In this platform, the production of spheroids with a diameter of 500 μm was realized in 7 days; this could not be produced with the traditional hanging-drop approach because of the limited exchange of the culture medium. Furthermore, the conventional hanging-drop technique limits the formation of embryoid bodies because of the intensive workload and difficulty in changing the culture medium manually. (137) To overcome these limitations, a PDMS-based microfluidic hanging-drop device containing microfluidic channels and wells was developed. (100) A mouse embryonic stem cell suspension containing 3 × 105 cells/mL was introduced in the channels under hydrostatic pressure. The cells were trapped in the wells of the microfluidic chip, and the 80–120 μm diameter of the embryoid body was easily formed in a 1-day cultivation. In another study, a pump-integrated PDMS microfluidic chip was designed to improve the flow control in the hanging-drop system. (136) The physiological pulsative flow was achieved using the pneumatically actuated pump. Human iPS cell-derived cardiac microtissue spheroids were developed using this system, and formed spheroids exhibited a beating at a rate of 60–90 bpm, which is similar to the natural frequency of the human heart. A microfluidic hanging-drop-based spheroid co-culture system was also used to facilitate the formation and co-culture of embryoid bodies and tumor spheroids. (98) In this study, spheroid generation up to 730 ± 27 μm was reported in Matrigel, and 16 spheroids per chip were produced in 10 days. (98)

3.5. Microstructures

The basic operation of this approach is based on delivering the cell suspension to the chip via a microfluidic channel and accumulating the cells in the microstructures patterned on the channel surface (Figure 2E). (138) The accumulated cells form spheroids, and the medium can be constantly fed into the chip via microchannels. (139) The diameters of the spheroids may be fine-tuned with the sizes and geometry of the microstructures. The major benefits of microstructures are that they protect cells from shear stress injury and allow delivery of nutrients to cells without damaging the spheroid structure. (106) In microstructure-based systems, the pressure generated by the flow rate in front of the miniature structures within the chip is critical, and this stagnation pressure also generates a stagnation zone within the microstructures. Thus, although the culture media flow at a constant rate from the outside region of the structures, the static environment created within the structures promotes cell fusion and the formation of spheroids. (140) Although there are not as many literature examples as the hanging-drop method and multiwell-based approach, there are promising examples using microstructures, which can be practically applied using photolithographic and molding approaches. (106−108,113,141) For example, LCC6 breast cancer cells encapsulated with alginate beads having a size of approximately 250 μm were trapped via a U-shaped microstructure for spheroid formation. The effect of doxorubicin on the produced spheroids was also studied on the same chip. (109) Furthermore, an in vitro breast tumor model containing an endothelial monolayer and ECM was reported on a chip to imitate a microvessel wall. The developed platform allowed the formation of uniformly sized multicellular tumor spheroids, and the platform was proposed further for use in drug-screening studies. After 14 days of culture, the average diameter of both BT549 and T47D breast cancer cell spheroids was determined to be around 180 μm, and formed spheroids showed good cell viability (>90%). (110) In another study, a Si-based microfluidic chip with pyramid-like microstructures was used to fabricate and culture spheroids. (107) With this device, 100 μm diameter MCF-7 breast cancer spheroids were produced in 2 days of culture, and spheroids’ sizes could be adjusted using different cell concentrations. A microfluidic device with U-shaped arrays was also utilized for a 3D cell culture of A549 cells under a continuous flow of culture medium. (112) With this device, 16 spheroids can be produced in 72 h.

3.6. External Forces

The formation of spheroids can also be established with external forces such as acoustic actuation (142) and dielectrophoresis (DEP). (143) The major hurdle in dealing with these systems is the possibility of the cell damage caused by mechanical stresses inside the system. Hence, it is difficult to establish long-lasting cell spheroids using these approaches. (23)

3.6.1. Acoustic Actuation

Acoustic-wave-based patterning can control the spatial position of cells. In this method, acoustic vibrations create a pressure gradient in a liquid, enabling simple, quick, noncontact, and precise 3D design of suspended cells in media or ECM-based hydrogels. (144) With use of acoustic node assembly, many different complex cellular patterns, as well as spheroids, and also their patterns, can be fabricated rapidly. (144,145) Low-frequency acoustic fields used in these systems prevent the heating of the solution and damage cells.
Acoustic waves allow label-free and contactless cell actuation in different fluidics. When compared to conventional spheroid formation methods, acoustic actuation (Figure 2F) allows higher throughput, easier size control, higher reproductivity/cell viability, and adaptability to different cell lines. (146) For example, a PDMS-based microfluidic platform containing 60 parallel microfluidic channels with dimensions of 150 μm height and width and a depth of 3 mm was integrated with a surface sound acoustic wave (SSAW) generator. (114) With this scheme, over 12,000 multicellular tumor spheroids can be generated in a few minutes. In another work, an acoustic-fluid device was used for the fabrication of homotypic and heterotypic spheroids without using any scaffold materials. (115) In the device, the acoustic radiation force acted on the suspended cells to collect them in pressure nodes, and the spheroid size and cell components could be easily adjusted by changing the initial cell concentration and ratio. The device allowed spheroids to form a diameter of 185.2 ± 50 μm in 9 h.

3.6.2. Dielectrophoresis

DEP in microfluidic platforms is a favorable method because of its fast, controllable, and efficient cell-patterning abilities. (134) DEP is an electrical force categorized as negative and positive DEP that can be used for spheroid formation (Figure 2G). (147) For instance, cell clusters were formed between the interdigitated electrodes using positive DEP. (116) The aggregates of Jurkat cells and AC3 mouse stromal cells were generated by using a potential difference of 20 V at 1 MHz frequency. The developed method was reported to be suitable for investigating the cellular interactions in 3D cell aggregation in different sizes by adjusting the magnitude of the electrical field applied between electrodes. In another study, a negative DEP device with 3D upper and lower interdigitated electrodes was used to form HuH7 human hepatoma cell aggregates within 45 min. (117)

4. Organ-on-a-Chip Applications Using Spheroids

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The integration of microfluidics and tissue engineering constitutes organ-on-a-chip (OOC) technologies. (148) OOC technologies enable biological systems to be created in a controllable manner under certain physiological conditions. Consequently, the imitation of in vivo microenvironment conditions can be achieved so that tissue and organ physiology may be generated under in vitro conditions. (149) Because of close contacts between cells and multicellular features, spheroids mimic paracellular signaling and physiological interface in heterogenic tissues or organs. Therefore, spheroids combined with OOCs can be a good candidate for accurate disease modeling. (150) Furthermore, in preclinical pharmacological studies, spheroids employed in OOC systems may show more realistic drug responses compared to conventional cell culture methods by elucidating in vivo physiological conditions. (151) Evolving OOC technology with spheroids provides researchers with several perspectives, especially in disease-modeling and drug-screening studies.

4.1. Disease Modeling

A disease model can be applied to animals or cells to exhibit all or a subset of both pathological processes and physiological functions found in corresponding human or animal illnesses. Examining disease models enables to gain a better understanding of how diseases arise and to evaluate new treatment methods. (152) An important advantage of disease models is that the accuracy of epidemiological assumptions can be validated with the mathematical description of biological processes, allowing us to gain deeper insight into the onset and course of diseases. (153) OOC systems could be utilized for disease modeling that could speed up the development of next-generation drugs and treatments for different diseases. (154)
Alzheimer’s disease is an age-related neurodegenerative disease that occurs because of amyloid-β accumulation. (155) The 2D cell culture studies conducted in this field remain incapable of understanding the pathogenesis, progression, and underlying reasons for Alzheimer’s disease. (156) In a study, a 3D brain model on a chip providing brain-like interstitial flow was developed to imitate a brain model with and without Alzheimer’s disease. (157) The neurospheroids obtained from neural progenitor cells were cultured in the presence and absence of amyloid-β on a single chip. In the end, low cell viability, high neural disorders, and dysfunction were reported in neurospheroids exposed to amyloid-β. Hence, the investigation of pathophysiological properties of Alzheimer’s in vitro was enabled with the 3D brain-on-a-chip.
Nonalcoholic fatty liver disease (NAFLD) is one of the most common chronic illnesses, caused by aberrant fat accumulation, decreased protein production, and deficiencies in numerous biological functions. (158) To investigate NAFLD pathogenesis, multicellular aggregates were prepared by using HepG2 and HUVECs. (159) The function of the hepatocytes was evaluated in a chip in terms of albumin secretion and reactive oxygen species level following the multicellular aggregates exposed to free fatty acids. On the basis of the obtained results, the implementation of a diet without fat and an antisteatotic drug can enable regression of NAFLD.
The communication between the liver and the pancreas is another essential mechanism for the human body to control insulin-glucose regulation. (160) OOC has great potential for investigating type-II diabetes mellitus in vitro. For instance, insulin secreted by islets positively affected glucose uptake from liver spheroids in the OOC system, and the glucose value in the medium was decreased. (161)
Ductal carcinoma in situ (DCIS) is a cancerous lesion formed by the collection of neuroplastic epithelial cells in the mammary duct. (162) To model an early stage of breast cancer with DCIS, human breast cancer-on-a-chip was developed. (163) For this purpose, breast cancer cell spheroids produced by a hanging-drop plate were collected and co-cultured with mammary ductal epithelial cells and fibroblasts to mimic the 3D structural organization of the human mammary duct. It was noted that the progression of malignancy of DCIS was successfully simulated via a compartmentalized 3D microfluidic device.
Inflammation and a variety of other disorders damage the integrity of endothelium and epithelial cellular barriers, which are critical for the selective transit of solutes and other molecules throughout the body. (164) A co-culture of 3D tumor spheroids and cellular barriers was demonstrated in a microfluidic chip containing multiple wells with HT29 spheroids and an MDCK cellular barrier. (165) The electrodes integrated into the chip enabled also transepithelial/transendothelial electrical resistance measurements to rapidly assess barrier sealing in real time.

4.2. Drug-Screening Studies

Typically, the drug development process is separated into three distinct phases: discovery, preclinical development, and clinical testing. The preclinical study is based on drug-screening research. (166) Drug screening is the process of identifying and optimizing prospective medicines before the selection of a candidate drug for clinical trials. (109) 3D cultures, particularly spheroids, have several benefits that have made them a popular tool for in vitro drug testing. (167)
For on-chip breast cancer drug delivery experiments, multicellular tumor spheroids (MCTSs) mimicking an ECM structure and monolayer endothelial cells mimicking a capillary channel structure were used. (110) MCTSs were created by using BT549 (triple-negative breast cancer) and T47D (nontriple negative breast cancer) cells. Doxorubicin (DOX) was used as an anticancer drug with carbon-dots-based nanocarriers utilized for drug delivery. Transportation of the drug between the vessel and the ECM and its penetration into the MCTSs were tracked in real time using breast cancer-on-a-chip. In addition, in situ cytotoxicity testing was also performed in a single chip, and it was observed that controlled distributed DOX caused higher cytotoxicity in BT549 spheroids than in T47D spheroids. Furthermore, the efficiency of different chemotherapy drugs was also tested on breast cancer spheroids in a microfluidic chip. (168,169) An evaluation of the efficacy of drugs cell viability analysis was conducted on bright-field micrographs of spheroids without using any staining process.
In a study based on vascularized cancer-on-a-chip, how the vascular structure of the tumor affected the drug resistance in tumor spheroids was investigated. (170) For this purpose, the obtained multicellular tumor spheroids composed of human lung fibroblasts (hLFs), MCF-7, and HUVECs with and without vasculature were exposed to paclitaxel (PTX)anticancer drug. The results indicated that whereas PTX inhibited cell growth in static culture, it did not have the same impact in dynamic culture because of perfusion-induced vascularization.
In another study, tumor-on-a-chip was developed for the investigation of the effects of the PTX-loaded liposomes on an ovarian cancer model. (171) The human ovarian cancer cell (SKOV3) spheroids formation, cultivation, and drug administration studies were conducted on the same chip. The four different formulations of liposomes were applied to the cells in 2D monolayer and 3D spheroids in different sizes under certain flow conditions. Consequently, small-sized tumor spheroids show better treatment efficacy with a low flow rate.
Temozolomide (TMZ) and bevacizumab (BEV) as clinical anticancer drugs were also tested for a high-grade and aggressive brain cancer model on a brain cancer chip containing glioblastoma multiform (GBM) spheroids. (172) The drugs were applied to the primary human-derived GBM tumor spheroids both alone and in combination. According to the results, the combination of TMZ and BEV exhibited high treatment efficiency compared to that of a single TMZ application. This study has shown that the efficient combination of chemotherapy drugs, namely, drug cocktails, can be rapidly determined using a brain cancer-on-a-chip-based system.
To inspect a topically used antifungal drug terbinafine, reconstructed human skin and liver spheroids were cultured separately using a two-compartment TissUse’ HUMIMIC Chip2. (173) The combination of skin and liver models enabled the measurement of toxic or metabolic reactions to the accumulated drug in the liver via transit through the skin barrier, which could not be assessed using conventional skin equivalents. In skin models, systemic terbinafine exposure boosted EGFR expression, whereas in the liver model on the same chip, it promoted apoptosis and decreased hepatic albumin expression.

5. Discussion

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5.1. Cells and Biomaterials Used for Spheroid Engineering

To obtain 3D spheroids in a microfluidic system, various types of cells have been used (Table 1). In addition, co-culture studies have also been performed by using at least two cells at once to produce multicellular spheroids. (174) There are several criteria to be considered in the evaluation of culture conditions according to used cell types in spheroid formation. (175) For a spheroid generation, the optimal cell number, composition of the culture medium, and culture period should be determined. For co-culture experiments, the ideal cell ratio should also be identified. Additionally, the cellular process, such as doubling time, differentiation, metabolism, and survival rate, needs to be considered carefully to form a holistic microtissue. (176) The success of the spheroid formation due to the cell types may differ. (177) For example, osteosarcoma cells, human umbilical vein endothelial cells (HUVECs), human glioblastoma cells, tumor epithelial cells (TEC), and mesenchymal stem cells have been shown to proliferate more rapidly in 3D cultures than breast cancer cells, sheep-derived bone marrow stem cells, rat interior tibialis muscle cells, and smooth muscle cells. (1) The size of formed spheroids could differ in the molecular characteristics of spheroids. For instance, HepG2 spheroids with a diameter of 200 μm showed higher albumin secretion compared to spheroids with a diameter of ≥300 μm. (178) Hence, the size of spheroids should be precisely determined to get desired cellular functionality.
Microfluidic fabrication provides different options for fabricating large or small spheroids depending on applications. (14,179−181) The type and density of the cells have been identified as the key issues concerning the diameter of the spheroids. (20) For example, 107 embryonic stem cells (ES)/mL, 107 HepG2 cells/mL, and 105 monkey kidney epithelial fibroblast (COS-7) cells/mL were fed into the microfluidic chip to generate spheroids. (182) Although ES and COS-7 cells formed uniform aggregates at 16 and 24 h, HepG2 cells exhibited irregular spheroid formation at 24 h. The average diameter of the spheroids formed by using ES and COS-7 was measured as 80 μm, whereas the spheroids of HepG2 were 200 μm in diameter in a 3-day culture. Hence, the same initial cell density can result in the development of spheroids of varying diameters depending on the cell type. On the other hand, the cell density may cause different spheroid shapes depending on the type of the cell. For example, although MG63 and HepG2 cells were loaded into the chip system with 8 × 106 cells/mL concentrations, MG63 cells showed a spherical shape, whereas HepG2 cells showed a nonspherical structure in a 5-day culture. Moreover, high cell density has the potential to clog microfluidic channels and result in the formation of nonspherical aggregates. (183)
Several matrixes have been used in spheroid-engineering research because of their biomimicry properties. (184) Although various matrixes are produced and used in various shapes and sizes for 3D culture, the matrixes to be used within the framework of spheroid engineering in microfluidic devices are favored in a hydrogel configuration because of their formable properties. (185) Natural polymers, synthetic polymers, and a variety of decellularized matrixes are the primary components utilized to create the hydrogel structure in this context. (186) Natural polymers are of great interest in terms of hydrogel preparation because of their excellent biocompatibility, biodegradability, nontoxic natures, and resemblance to the form of native ECM. (187) However, being physically weak and having components activating the immune or inflammatory response can discredit these materials. (187) Thanks to their adjustable mechanical properties, biodegradation, and cross-linking density, the usage of synthetic polymers is more desirable than natural polymers. (188) However, considerable attention should be paid to the problem of toxicity and biocompatibility that may arise from the reagents used in the synthesis phase. (189) Apart from the polymers, decellularized-matrixes-derived hydrogels have been proposed for 3D culture studies on the basis of their site-specific biochemical and mechanical cues, the capability of regulating cellular behavior (e.g., attachment, proliferation, migration, and differentiation), and excellent resemblance to native tissue ECM. (190) Nevertheless, several issues, such as loss of mechanical properties, pathogen transmission risk, deteriorated ECM structure or functionality, and immunogenic problems, need to be overcome. (191) Because of these reasons, these materials have been utilized in combination rather than alone (Table 1). Hence, bringing these materials together can compensate and strengthen the properties of each material.

5.2. Design Parameters of Microfluidic Systems for Spheroid Engineering

The major goal of employing microfluidics in spheroid engineering is to transport cells to a predefined position on a chip while maintaining a continuous flow of culture media within the chip as the spheroid forms and maintains. (192) The developed microfabrication techniques enable fabrication of complex and physiologically suitable microstructures in microfluidic chips that can affect the stability of the spheroid formation and size. (193) For example, MPM H2052 cells were cultured in microchannels, including round-bottom and flat-bottom microwells. (194) Because of the influence of the shape of the microstructure, round-bottom microwells have been reported to provide higher spheroid-forming efficiency than flat-bottom microwells. On the other hand, the symmetrical treelike structure employed in the design guaranteed that the hanging cells were retained homogeneously in eight microwells with the effect of gravity, resulting in the homogeneous size distribution of the spheroids created.
The advantages and limitations of spheroid fabrication techniques used in the microfluidic devices are summarized in Table 2. Microwell and droplet-based spheroid fabrication methods are widely used in microfluidic systems (Table 1). The main reason for this may be that droplet-based systems can produce thousands of spheroids in minutes or even seconds. Moreover, microwell-based systems do not require sophisticated lithographic manufacturing procedures, so they are cost-effective and practical systems. Additionally, microwell-based systems could be used to fabricate spheroids in different size ranges. (195) Despite the growing interest in microfluidic hanging-drop and microstructure approaches for spheroid manufacturing, there are significant restrictions that preclude their utilization. For example, it is extremely difficult to establish a stagnation zone on the chip’s microstructures that do not harm cells. (196) For this purpose, flow rate and shear stress in the chip must be controlled precisely to form and maintain the spheroids. (196) Additionally, the recovery of spheroids produced by these technologies can be quite challenging. (20) In the microfluidic hanging-drop technique, the nonuniform distribution of cells in each droplet can result in size and shape variations in formed spheroids. (101) Additionally, one of the most fundamental issues with the microfluidic hanging-drop approach is that the culture medium cannot be replaced dynamically not to deteriorate the droplet structure. (101) Electrowetting, acoustic, and dielectrophoresis methods are the least used techniques for spheroid fabrication. Electrowetting is limited in use because of challenges in designing and fabricating these electrowetting platforms. (125) In spite of its advantages, such as flexible liquid handling and label-free manipulation, the acoustic method has downsides like contamination difficulties at the liquid–liquid interface and undesired heating issues. (127) Dielectrophoresis also has tremendous potential for quick and precise cell manipulation. (197,198) Nonetheless, with this approach, particular attention should be paid to the inability to establish cellular interactions due to the high electrical conductivity of the culture medium, as well as the risk of cell injury due to the strong electrical field. (199,200) Although magnetic-based techniques were not utilized for spheroid fabrication in microfluidic channels, they have the potential to be used for fabricating self-assembled spheroids in a label-free manner. (42,201)
Table 2. Comparison of the Microfluidic-Based Methods for Spheroid Fabrication
methodsadvantageslimitations
droplet-based• create identical templates for spheroid formation• resulting empty droplets (no cell containment)
 • single, double, and triple encapsulation variations• insufficient nutrient supply
electrowetting• easy automation and integration• hard to design and fabricate these platforms
 • rapid analysis 
 • pump and valve-free operation 
microwell• simple to operate• cell loss and spheroid disruption during spheroid collection
 • controllable spheroid size 
microfluidic hanging drop• self-assembly due to gravity• high flow rate used to collect formed spheroids can damage spheroids
 • no cell adhesion observed on the surfaces• nonhomogeneous number of cells in each hanging drop
microstructures• reversible process enabling formation and collection of spheroids• applying high flow rate can affect the spheroid formation time and make cells escape
 • efficient cell trapping due to high cellular interaction 
acoustic• rapid spheroid formation enabling high cell viability• possible cell damage due to heating problems while using high-frequency acoustic fields
 • simple and versatile technology to fabricate complex spheroids patterns in mild conditions• complex fabrication processes while integrating acoustic wave generators on chip level
dielectrophoresis• fast cell manipulation• possible cell damage due to high electrical field
 • stable cell positioning• high conductivity of culture medium may result in low cellular interactions and induce cell damage
Researchers have highlighted the necessity of a “well-controlled environment” and “more in vivo like circumstances” as advantages of microfluidic-based approaches over conventional procedures. However, when the studies presented in this review article are evaluated in this context, it is evident that the vast majority of them have focused on producing high-quality and large numbers of spheroids rapidly rather than providing a well-controlled microenvironment for the long-term culture of spheroids. Only a few reports provide objective information on these two challenges. (61,82,98,102)In vivo like conditions can imply the physical conditions of cells in an organism that can simulate the 3D cellular structure and its microenvironment. (202,203) Because of mimicking the multicellular structure and ECM synthesis, spheroids can provide more realistic in vivo conditions than a 2D cell culture. (27) Furthermore, the studies show that spheroids possess a level of protein and gene expressions, which are similar to those in vivo. (14) The cellular microenvironment, on the other hand, is a very dynamic and complex structure both biomechanically and biochemically and consists of ECM, fluid flow, biomolecular gradients, and other cell types. (204) Although it is quite challenging to imitate such a complex structure in vitro, it is necessary to obtain physiologically more realistic 3D structures consisting of well-defined spatial and temporally controlled cells. (205) When the studies presented in this review article are evaluated in this context, few articles used biomaterials, such as alginate, PEG, and collagen, to better mimic the biomechanical and biochemical microenvironment in spheroid cultures composed of cancer/epithelial/endothelial cells. (65,67,72,73,75,77,88,92,106) These biomaterials can provide enhanced cell–cell and cell–ECM interactions. (19,184) Moreover, co-cultured cells could be arranged spatially and temporally in spheroids with controlled perfusion. (61,82,98,102)In vivo systems are already quite complex, and thus the full mimicry of these systems is still very challenging now. However, organ-on-a-chip technologies used for the spheroid culture that provide various advantages compared to 2D culture could further develop in terms of biomaterial, perfusion, and co-culture perspective to get closer results to those obtained in in vivo conditions.
To mimic the physiological conditions better in vitro, there should be several design criteria, such as shear force, medium delivery, chip architecture, and cell type and density, that need to be considered in OOC applications containing spheroids. In the OOC system, nutrient distribution, waste removal, and transport of molecules are created by liquid flow. OOC with liquid shear stress provides better biological function and capability compared to static culture systems. (206,207) Shear stress can also affect the cell cycle, cell differentiation, gene expression, and signaling of molecules in tumor cells. (208,209) Moreover, the cells with a high tendency to cluster were reported to form spheroids under high shear force. (96)
To simulate the circulation of the vascular tissue, continuous circulation of the fresh culture medium is desired for OOC applications. (210) There have been several methods proposed for the homogeneous distribution of the culture medium, such as surface-tension-driven flow, syringe and peristaltic pumping, and hydrostatic and osmotic pressure difference. (211) Hydrostatic pressure is generated with the help of the pressure difference between the inlet and the outlet on a chip. This method provides a suitable medium exchange for a perfusion cell culture system. (212) Osmotic pumping is based on the usage of permeable membrane and driving agents in different concentrations. (211) For instance, the perfusion was ensured with polyethylene glycol (PEG) concentration in a controlled manner by using a cellulose membrane for hepatocyte spheroid formation. (213) On the other hand, syringe pumping has widely been used for perfusion in spheroid-engineering studies as it enables controllable and continuous flow. (194,214) A peristaltic pump is an active pumping system that enables liquid manipulation with a positive displacement of a flexible conduit. (211) For example, with use of a peristaltic pump, the constant medium flow was generated for the perfusion of hepatic spheroids. (215) Pumping systems that have different working principles enable continuous medium circulation in a cell culture, but hydrodynamic and osmotic pumping systems have several limitations, for instance, the requirement of conductive reagents, low flow rate, and pressure. However, syringe and peristaltic pumping systems permit a sufficient flow rate, simple integration, easy control, and rapid response time. (216)

5.3. Current Challenges and Future Perspectives

First, chip material is an important element for spheroid formation and OOC applications. Polydimethylsiloxane (PDMS) is the most frequently used material in the fabrication of microfluidic chips because of its low cost, ease of use, transparency, elasticity, biocompatibility, and gas-permeable properties. (217) In addition, glass-based materials can be used for fabricating microfluidic devices with their optical transparency, chemical inertness, rigidity, and high-temperature resistance features. However, they are insufficient for mass production because of the slow and expensive production process. Hence, new materials can be exploited for spheroid fabrication and its applications. Furthermore, the fabrication of microfluidic chips should be simple and cost-effective to increase microfluidic chip usage. Herein, 3D printing technologies that allow rapid, easy, and low-cost production can be used for the fabrication of microfluidic systems from different materials. These technologies also enable fabrication of transparent and complex geometries in a short time. (218,219) Meanwhile, design parameters such as fluid shear force, concentration gradient, and dynamic mechanical stress should be improved while mimicking physiological conditions in vitro using integrated elements such as pumps, valves, and different actuators. In addition, the integration of these systems can enable automated, highly efficient, and reproducible experiments. Different sensors can also be integrated into the microfluidic chips to monitor different parameters to evaluate spheroid formation and culture in real time. Integrated sensor systems can be used not only to detect the amount of metabolic product in the culture environment but also to simultaneously observe cell behavior, mechanical and electrical stimulation, chemical gradient, pH, and gas changes. (220) Therefore, the spheroid formation and culture process can be easily controlled and also be adjusted for repeatable experiments. Furthermore, these sensors can be utilized to monitor drug responses of formed spheroids. (221)
Furthermore, an all-encompassing culture medium should be determined to culture spheroids containing more than one cell type. To model an organ truly in vitro, the metabolites and microbiome system of this organ need to be integrated on a chip level with spheroid culture. Instead of using cell lines, patient-specific cells can be used to form spheroids for personalized medicine applications. Patient-derived spheroids can be utilized in microfluidic chips to assess drug efficacies for each individual that can be employed for digital twin applications. While constituting these chips, one should also consider the differences between the individuals, such as age, gender, ethnicity, and genetics.
Lastly, the developments in OOC technology can eliminate animal testing in preclinical studies and enable multiple drug testing on a single chip. Similarly, the integration of emerging “artificial intelligence” techniques into microfluidic-based spheroid-engineering methods by controlling spheroid size formation, evaluating new drug candidates, and analyzing drug responses could lead to the development of next-generation tools for effective drug studies. (168,222) These developments can eliminate animal tests and accelerate preclinical analyses and may lead to “clinical trials-on-a-chip” technology in the future.
The global 3D cell culture market including spheroid engineering is expected to grow from $1.3 billion (U.S. dollars) in 2022 to $2.6 billion by 2027, at a compounded annual growth rate (CAGR) of 15.6% between 2022 and 2027. (223) Conventional spheroid-engineering methodologies are applied in different products using 24-, 96-, or 384-well microtiter plates, such as Perfecta3D hanging drop plates, (224) Corning spheroid ULA (Ultra-Low Attachment surface) microplates, (225) and MicroTissues 3D Petri Dish micromold spheroids. (226) Moreover, droplet-based 3D bioprinted technology could also be applied for spheroid fabrication. (227,228) There are also many companies in the market supplying solutions for microfluidic-based 3D cell culture and organ-on-a-chip. For instance, Fluigent has developed microfluidic chips that are used in 3D cell culture to generate an hypoxia environment for tumor cells, endothelium/epithelium barrier, and vascularization. (227) Creative Biolabs Microfluidics Company supplies 3D cell culture chips. These chips enable a culture of neurons, skin, stomach, intestinal, and kidney cells and also their co-culture. (227) In addition to these companies, Emulate has improved organ-specific organ-on-a-chip devices for modeling of brain, colon intestine, duodenum intestine, kidney, liver, and lung. (227) Moreover, Dolomite Microfluidics, (227) Schott Minifab, (227) and Droplet Genomics (227) developed droplet-based and microwell-based microfluidic devices. These commercial devices could possibly be applied for spheroid-engineering applications.

6. Conclusion

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2D culture systems with monolayer cells are limited by cell–cell and cell–extracellular matrix interactions, and these systems cannot mimic the cell microenvironment. However, 3D culture systems overcome these limitations and equally provide nutrient, gas, and growth factor transport into cells. Spheroids as 3D cell aggregates show similar functions to in vivo tissues. These cell aggregates are traditionally fabricated with various techniques such as pellet culture, hanging drop, spinner culture, magnetic levitation, etc. Microfluidic approaches have also been developed for spheroid studies. Compared to conventional spheroid formation methods, microfluidic systems ensure the controlled formation of a spheroid with simple and automated processing steps. Moreover, using spheroids in OOC models allows the easy realization of in vivo conditions for disease-modeling and drug-screening fields to understand complex physiologies of tissues and organs. In the field of OOC, studies composed of the production of single tissues or organs have come a long way in the past decade. Especially, the co-culture of different cell types on a single chip can simulate better in vivo physiological conditions. However, for multiple tissue studies, a different cell culture medium was necessary for different cell lines. To overcome this bottleneck, the spheroid culture of different cells may be maintained in different microcompartments on a chip, and sensors and actuators may also be integrated to monitor and regulate metabolites in a culture medium. Hence, these developments in spheroid engineering using microfluidics could lead to next-generation tools for accurate disease modeling and treatment.

Data Availability

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The data underlying this study are available from the corresponding author upon reasonable request.

Author Information

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  • Corresponding Author
  • Authors
  • Author Contributions

    A.T. and S.K. contributed equally to this article.

    Author Contributions

    H.K. and H.C.T. designed the content of the article; all authors performed literature survey and wrote the article. A.T., S.K., and O.S.O. prepared the figures and tables. H.K. and H.C.T. edited and reviewed the article.

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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Financial support by The Scientific and Technological Research Council of Turkey for the 119M052 (H.C.T.) grant is gratefully acknowledged. H.C.T. is thankful for the Outstanding Young Scientists Award funding (TUBA GEBIP 2020) from the Turkish Academy of Science and Young Scientist Awards (BAGEP 2022) from Science Academy (Bilim Akademisi). S.K. acknowledges the support of the Turkish Council of Higher Education for a 100/2000 CoHE doctoral scholarship. Authors thank Engin Ozcivici, Ph.D., from Izmir Institute of Technology, Department of Bioengineering, for valuable discussions.

References

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    Kim, S.-j.; Kim, E. M.; Yamamoto, M.; Park, H.; Shin, H. Engineering Multi-Cellular Spheroids for Tissue Engineering and Regenerative Medicine. Adv. Healthc. Mater. 2020, 9 (23), 2000608,  DOI: 10.1002/adhm.202000608
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    17
    Engineering multi-cellular spheroids for tissue engineering and regenerative medicine
    Kim, Se-jeong; Kim, Eun Mi; Yamamoto, Masaya; Park, Hansoo; Shin, Heungsoo
    Advanced Healthcare Materials (2020), 9 (23), 2000608CODEN: AHMDBJ; ISSN:2192-2640. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A review. Multi-cellular spheroids are formed as a 3D structure with dense cell-cell/cell-extracellular matrix interactions, and thus, have been widely utilized as implantable therapeutics and various ex vivo tissue models in tissue engineering. In principle, spheroid culture methods maximize cell-cell cohesion and induce spontaneous cellular assembly while minimizing cellular interactions with substrates by using phys. forces such as gravitational or centrifugal forces, protein-repellent biomaterials, and micro-structured surfaces. In addn., biofunctional materials including magnetic nanoparticles, polymer microspheres, and nanofiber particles are combined with cells to harvest composite spheroids, to accelerate spheroid formation, to increase the mech. properties and viability of spheroids, and to direct differentiation of stem cells into desirable cell types. Biocompatible hydrogels are developed to produce microgels for the fabrication of size-controlled spheroids with high efficiency. Recently, spheroids have been further engineered to fabricate structurally and functionally reliable in vitro artificial 3D tissues of the desired shape with enhanced specific biol. functions. This paper reviews the overall characteristics of spheroids and general/advanced spheroid culture techniques. Significant roles of functional biomaterials in advanced spheroid engineering with emphasis on the use of spheroids in the reconstruction of artificial 3D tissue for tissue engineering are also thoroughly discussed.
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  18. 18
    Kamatar, A.; Gunay, G.; Acar, H. Natural and Synthetic Biomaterials for Engineering Multicellular Tumor Spheroids. Polymers (Basel). 2020, 12 (11), 2506,  DOI: 10.3390/polym12112506
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    18
    Natural and synthetic biomaterials for engineering multicellular tumor spheroids
    Kamatar, Advika; Gunay, Gokhan; Acar, Handan
    Polymers (Basel, Switzerland) (2020), 12 (11), 2506CODEN: POLYCK; ISSN:2073-4360. (MDPI AG)
    A review. The lack of in vitro models that represent the native tumor microenvironment is a significant challenge for cancer research. Two-dimensional (2D) monolayer culture has long been the std. for in vitro cell-based studies. However, differences between 2D culture and the in vivo environment have led to poor translation of cancer research from in vitro to in vivo models, slowing the progress of the field. Recent advances in three-dimensional (3D) culture have improved the ability of in vitro culture to replicate in vivo conditions. Although 3D cultures still cannot achieve the complexity of the in vivo environment, they can still better replicate the cell-cell and cell-matrix interactions of solid tumors. Multicellular tumor spheroids (MCTS) are three-dimensional (3D) clusters of cells with tumor-like features such as oxygen gradients and drug resistance, and represent an important translational tool for cancer research. Accordingly, natural and synthetic polymers, including collagen, hyaluronic acid, Matrigel, polyethylene glycol (PEG), alginate and chitosan, have been used to form and study MCTS for improved clin. translatability. This review evaluates the current state of biomaterial-based MCTS formation, including advantages and disadvantages of the different biomaterials and their recent applications to the field of cancer research, with a focus on the past five years.
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  19. 19
    Vadivelu, R. K.; Kamble, H.; Shiddiky, M. J. A.; Nguyen, N. T. Microfluidic Technology for the Generation of Cell Spheroids and Their Applications. Micromachines. 2017, 8, 94,  DOI: 10.3390/mi8040094
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    Moshksayan, K.; Kashaninejad, N.; Warkiani, M. E.; Lock, J. G.; Moghadas, H.; Firoozabadi, B.; Saidi, M. S.; Nguyen, N. T. Spheroids-on-a-Chip: Recent Advances and Design Considerations in Microfluidic Platforms for Spheroid Formation and Culture. Sensors and Actuators, B: Chemical. 2018, 263, 151,  DOI: 10.1016/j.snb.2018.01.223
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    20
    Spheroids-on-a-chip: Recent advances and design considerations in microfluidic platforms for spheroid formation and culture
    Moshksayan, Khashayar; Kashaninejad, Navid; Warkiani, Majid Ebrahimi; Lock, John G.; Moghadas, Hajar; Firoozabadi, Bahar; Saidi, Mohammad Said; Nguyen, Nam-Trung
    Sensors and Actuators, B: Chemical (2018), 263 (), 151-176CODEN: SABCEB; ISSN:0925-4005. (Elsevier B.V.)
    A cell spheroid is a three-dimensional (3D) aggregation of cells. Synthetic, in-vitro spheroids provide similar metab., proliferation, and species concn. gradients to those found in-vivo. For instance, cancer cell spheroids have been demonstrated to mimic in-vivo tumor microenvironments, and are thus suitable for in-vitro drug screening. The first part of this paper discusses the latest microfluidic designs for spheroid formation and culture, comparing their strategies and efficacy. The most recent microfluidic techniques for spheroid formation utilize emulsion, microwells, U-shaped microstructures, or digital microfluidics. The engineering aspects underpinning spheroid formation in these microfluidic devices are therefore considered. In the second part of this paper, design considerations for microfluidic spheroid formation chips and microfluidic spheroid culture chips (μSFCs and μSCCs) are evaluated with regard to key parameters affecting spheroid formation, including shear stress, spheroid diam., culture medium delivery and flow rate. This review is intended to benefit the microfluidics community by contributing to improved design and engineering of microfluidic chips capable of forming and/or culturing three-dimensional cell spheroids.
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  21. 21
    Coluccio, M. L.; Perozziello, G.; Malara, N.; Parrotta, E.; Zhang, P.; Gentile, F.; Limongi, T.; Raj, P. M.; Cuda, G.; Candeloro, P.; Di Fabrizio, E. Microfluidic Platforms for Cell Cultures and Investigations. Microelectron. Eng. 2019, 208, 14– 28,  DOI: 10.1016/j.mee.2019.01.004
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    Microfluidic platforms for cell cultures and investigations
    Coluccio, Maria Laura; Perozziello, Gerardo; Malara, Natalia; Parrotta, Elvira; Zhang, Peng; Gentile, Francesco; Limongi, Tania; Raj, Pushparani Michael; Cuda, Gianni; Candeloro, Patrizio; Di Fabrizio, Enzo
    Microelectronic Engineering (2019), 208 (), 14-28CODEN: MIENEF; ISSN:0167-9317. (Elsevier B.V.)
    This review covers several aspects of microfluidic devices used for culturing and monitoring of both adherent and non-adherent cells, including a multitude of applications. A comparison of available platforms with high throughput anal., automation capability, interface to sensors and integration, is reported. Aspects, such as operational versatility of the devices, are scrutinized in terms of their anal. efficacy. It is found that due to multi-functionality capability of modern microfluidics, there is big amt. of exptl. data obtainable from a single device, allowing complex exptl. control and efficient data correlation, particularly important when biomedical studies are considered. Hence several examples on cell culture and monitoring are given in this review, including details on design of microfluidic devices with their distinctive technol. peculiarities.
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  22. 22
    Shao, C.; Chi, J.; Zhang, H.; Fan, Q.; Zhao, Y.; Ye, F. Development of Cell Spheroids by Advanced Technologies. Advanced Materials Technologies. 2020, 5, 2000183,  DOI: 10.1002/admt.202000183
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    Development of Cell Spheroids by Advanced Technologies
    Shao, Changmin; Chi, Junjie; Zhang, Hui; Fan, Qihui; Zhao, Yuanjin; Ye, Fangfu
    Advanced Materials Technologies (Weinheim, Germany) (2020), 5 (9), 2000183CODEN: AMTDCM; ISSN:2365-709X. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A review. 3D cell culture has been strongly advocated as a highly useful culture technique instead of the traditional 2D cell culture. Specially, spheroids, as the primary mode of 3D cell culture, allow cells to establish a connection between the cell and the extracellular matrix to form a specific 3D structure that better mimics the growing environment of cells in vivo. Benefiting from the emergence of various advanced micromachining platforms, spheroids have received increasing attention in diverse fields. Herein, the combination of the cell spheroid culture with microfluidic technol. is reviewed. After concisely introducing the traditional methods for fabricating cell spheroids, an outline of the approaches for prepg. cell spheroids using different advanced techniques is given. Then, the various applications of the generated cell spheroids in the field of biomedical engineering are presented and the current limitations, challenges, and future directions are summarized.
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    Shen, H.; Cai, S.; Wu, C.; Yang, W.; Yu, H.; Liu, L. Recent Advances in Three-Dimensional Multicellular Spheroid Culture and Future Development. Micromachines 2021, 12 (1), 96,  DOI: 10.3390/mi12010096
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    Hoarau-Véchot, J.; Rafii, A.; Touboul, C.; Pasquier, J. Halfway between 2D and Animal Models: Are 3D Cultures the Ideal Tool to Study Cancer-Microenvironment Interactions?. International Journal of Molecular Sciences. 2018, 19, 181,  DOI: 10.3390/ijms19010181
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    Halfway between 2D and animal models: are 3D cultures the ideal tool to study cancer-microenvironment interactions
    Hoarau-Vechot, Jessica; Rafii, Arash; Touboul, Cyril
    International Journal of Molecular Sciences (2018), 19 (1), 181/1-181/24CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)
    A review. An area that has come to be of tremendous interest in tumor research in the last decade is the role of the microenvironment in the biol. of neoplastic diseases. The tumor microenvironment (TME) comprises various cells that are collectively important for normal tissue homeostasis as well as tumor progression or regression. Seminal studies have demonstrated the role of the dialogue between cancer cells (at many sites) and the cellular component of the microenvironment in tumor progression, metastasis, and resistance to treatment. Using an appropriate system of microenvironment and tumor culture is the first step towards a better understanding of the complex interaction between cancer cells and their surroundings. Three-dimensional (3D) models have been widely described recently. However, while it is claimed that they can bridge the gap between in vitro and in vivo, it is sometimes hard to decipher their advantage or limitation compared to classical two-dimensional (2D) cultures, esp. given the broad no. of techniques used. We present here a comprehensive of the different 3D methods developed recently, and, secondly, we discuss the pros and cons of 3D culture compared to 2D when studying interactions between cancer cells and their microenvironment.
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    Lv, D.; Hu, Z.; Lu, L.; Lu, H.; Xu, X. Three-Dimensional Cell Culture: A Powerful Tool in Tumor Research and Drug Discovery. Oncology Letters. 2017,  DOI: 10.3892/ol.2017.7134
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    Oliveira, M. B.; Neto, A. I.; Correia, C. R.; Rial-Hermida, M. I.; Alvarez-Lorenzo, C.; Mano, J. F. Superhydrophobic Chips for Cell Spheroids High-Throughput Generation and Drug Screening. ACS Appl. Mater. Interfaces 2014, 6, 9488,  DOI: 10.1021/am5018607
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    26
    Superhydrophobic Chips for Cell Spheroids High-Throughput Generation and Drug Screening
    Oliveira, Mariana B.; Neto, Ana I.; Correia, Clara R.; Rial-Hermida, Maria Isabel; Alvarez-Lorenzo, Carmen; Mano, Joao F.
    ACS Applied Materials & Interfaces (2014), 6 (12), 9488-9495CODEN: AAMICK; ISSN:1944-8244. (American Chemical Society)
    We suggest the use of biomimetic superhydrophobic patterned chips produced by a benchtop methodol. as low-cost and waste-free platforms for the prodn. of arrays of cell spheroids/microtissues by the hanging drop methodol. Cell spheroids have a wide range of applications in biotechnol. fields. For drug screening, they allow studying 3D models in structures resembling real living tissues/tumors. In tissue engineering, they are suggested as building blocks of bottom-up fabricated tissues. We used the wettability contrast of the chips to fix cell suspension droplets in the wettable regions and evaluated on-chip drug screening in 3D environment. Cell suspensions were patterned in the wettable spots by three distinct methods: (1) by pipetting the cell suspension directly in each individual spot, (2) by the continuous dragging of a cell suspension on the chip, and (3) by dipping the whole chip in a cell suspension. These methods allowed working with distinct throughputs and degrees of precision. The platforms were robust, and we were able to have static or dynamic environments in each droplet. The access to cell culture media for exchange or addn./removal of components was versatile and opened the possibility of using each spot of the chip as a mini-bioreactor. The platforms' design allowed for samples visualization and high-content image-based anal. on-chip. The combinatorial anal. capability of this technol. was validated by following the effect of doxorubicin at different concns. on spheroids formed using L929 and SaOs-2 cells.
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    Lin, R. Z.; Chang, H. Y. Recent Advances in Three-Dimensional Multicellular Spheroid Culture for Biomedical Research. Biotechnology Journal. 2008, 3, 1172,  DOI: 10.1002/biot.200700228
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    Recent advances in three-dimensional multicellular spheroid culture for biomedical research
    Lin, Ruei-Zhen; Chang, Hwan-You
    Biotechnology Journal (2008), 3 (9-10), 1172-1184CODEN: BJIOAM; ISSN:1860-6768. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A review. Many types of mammalian cells can aggregate and differentiate into 3-D multicellular spheroids when cultured in suspension or a nonadhesive environment. Compared to conventional monolayer cultures, multicellular spheroids resemble real tissues better in terms of structural and functional properties. Multicellular spheroids formed by transformed cells are widely used as a vascular tumor models for metastasis and invasion research and for therapeutic screening. Many primary or progenitor cells show significantly enhanced viability and functional performance when grown as spheroids. Multicellular spheroids in this aspect are ideal building units for tissue reconstruction. Here the authors review the current understanding of multicellular spheroid formation mechanisms, their biomedical applications, and recent advances in spheroid culture, manipulation, and anal. techniques.
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  28. 28
    Kyffin, J. A.; Cox, C. R.; Leedale, J.; Colley, H. E.; Murdoch, C.; Mistry, P.; Webb, S. D.; Sharma, P. Preparation of Primary Rat Hepatocyte Spheroids Utilizing the Liquid-Overlay Technique. Curr. Protoc. Toxicol. 2019,  DOI: 10.1002/cptx.87
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    Achilli, T. M.; Meyer, J.; Morgan, J. R. Advances in the Formation, Use and Understanding of Multi-Cellular Spheroids. Expert Opinion on Biological Therapy. 2012, 12, 1347,  DOI: 10.1517/14712598.2012.707181
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    Advances in the formation, use and understanding of multi-cellular spheroids
    Achilli, Toni-Marie; Meyer, Julia; Morgan, Jeffrey R.
    Expert Opinion on Biological Therapy (2012), 12 (10), 1347-1360CODEN: EOBTA2; ISSN:1471-2598. (Informa Healthcare)
    A review. Introduction: Developing in vitro models for studying cell biol. and cell physiol. is of great importance to the fields of biotechnol., cancer research, drug discovery, toxicity testing, as well as the emerging fields of tissue engineering and regenerative medicine. Traditional two-dimensional (2D) methods of mammalian cell culture have several limitations and it is increasingly recognized that cells grown in a three-dimensional (3D) environment more closely represent normal cellular function due to the increased cell-to-cell interactions, and by mimicking the in vivo architecture of natural organs and tissues.Areas covered: In this review, we discuss the methods to form 3D multi-cellular spheroids, the advantages and limitations of these methods, and assays used to characterize the function of spheroids. The use of spheroids has led to many advances in basic cell sciences, including understanding cancer cell interactions, creating models for drug discovery and cancer metastasis, and they are being investigated as basic units for engineering tissue constructs. As so, this review will focus on contributions made to each of these fields using spheroid models.Expert opinion: Multi-cellular spheroids are rich in biol. content and mimic better the in vivo environment than 2D cell culture. New technologies to form and analyze spheroids are rapidly increasing their adoption and expanding their applications.
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  30. 30
    Marrella, A.; Fedi, A.; Varani, G.; Vaccari, I.; Fato, M.; Firpo, G.; Guida, P.; Aceto, N.; Scaglione, S. High Blood Flow Shear Stress Values Are Associated with Circulating Tumor Cells Cluster Disaggregation in a Multi-Channel Microfluidic Device. PLoS One 2021, 16 (1), e0245536,  DOI: 10.1371/journal.pone.0245536
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    30
    High blood flow shear stress values are associated with circulating tumor cells cluster disaggregation in a multi-channel microfluidic device
    Marrella, Alessandra; Fedi, Arianna; Varani, Gabriele; Vaccari, Ivan; Fato, Marco; Firpo, Giuseppe; Guida, Patrizia; Aceto, Nicola; Scaglione, Silvia
    PLoS One (2021), 16 (1), e0245536CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
    Circulating tumor cells (CTCs) can flow the bloodstream as single cells or as multicellular aggregates (clusters), which present a different potential to metastasize. The effects of the bloodstream-related phys. constraints, such as hemodynamic wall shear stress (WSS), on CTC clusters are still unclear. Therefore, we developed, upon theor. and CFD modeling, a new multichannel microfluidic device able to simultaneously reproduce different WSS characterizing the human circulatory system, where to analyze the correlation between SS and CTC clusters behavior. Three physiol. WSS levels (i.e. 2, 5, 20 dyn/cm2) were generated, reproducing values typical of capillaries, veins and arteries. As first validation, triple-neg. breast cancer cells (MDA-MB-231) were injected as single CTCs showing that higher values of WSS are correlated with a decreased viability. Next, the SS-mediated disaggregation of CTC clusters was computationally investigated in a vessels-mimicking domain. Finally, CTC clusters were injected within the three different circuits and subjected to the three different WSS, revealing that increasing WSS levels are assocd. with a raising clusters disaggregation after 6 h of circulation. These results suggest that our device may represent a valid in vitro tool to carry out systematic studies on the biol. significance of blood flow mech. forces and eventually to promote new strategies for anticancer therapy.
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  31. 31
    Fattahi, P.; Rahimian, A.; Slama, M. Q.; Gwon, K.; Gonzalez-Suarez, A. M.; Wolf, J.; Baskaran, H.; Duffy, C. D.; Stybayeva, G.; Peterson, Q. P.; Revzin, A. Core-Shell Hydrogel Microcapsules Enable Formation of Human Pluripotent Stem Cell Spheroids and Their Cultivation in a Stirred Bioreactor. Sci. Rep. 2021,  DOI: 10.1038/s41598-021-85786-2
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    Phelan, M. A.; Gianforcaro, A. L.; Gerstenhaber, J. A.; Lelkes, P. I. An Air Bubble-Isolating Rotating Wall Vessel Bioreactor for Improved Spheroid/Organoid Formation. Tissue Eng. - Part C Methods 2019, 25, 479,  DOI: 10.1089/ten.tec.2019.0088
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    An Air Bubble-Isolating Rotating Wall Vessel Bioreactor for Improved Spheroid/Organoid Formation
    Phelan, Michael A.; Gianforcaro, Anthony L.; Gerstenhaber, Jonathan A.; Lelkes, Peter I.
    Tissue Engineering, Part C: Methods (2019), 25 (8), 479-488CODEN: TEPCAE; ISSN:1937-3384. (Mary Ann Liebert, Inc.)
    Rotating wall vessel (RWV) bioreactors have been used to produce cell spheroids and organoids at a faster rate than in other bioreactor devices and with higher structural and functional fidelity. One of the limitations of traditional RWV systems is the well-documented tendency for air bubble formation during operation. The presence of these bubbles negates key features of the RWV environment, such as zero headspace, low-shear, and simulated microgravity. In this article, we describe the design, construction, and testing of a novel RWV capable of constantly removing air bubbles from the system without interfering with the fluid dynamics that produce optimized cell culture conditions. We modeled this capacity using computational fluid dynamics and then validated the model with alginate beads and spheroid cultures of A549 human lung adenocarcinoma cells. The areas of spheroids assembled from A549 cells in the novel bioreactor in the presence of air bubbles were an order of magnitude larger than in conventional bioreactors when bubbles were present. Our results demonstrate the ability of the novel design to remove and isolate bubbles while avoiding damage to spheroid assembly, as obsd. in conventional RWV bioreactors in the presence of bubbles. We anticipate that the novel design will increase exptl. reproducibility and consistency when using RWV bioreactors. Impact Statement : The rotating wall vessel (RWV) bioreactor is a powerful tool for the generation of sizeable, faster-growing organoids. However, the ideal, low-shear, modeled microgravity environment in the RWV is frequently disrupted by the formation of bubbles, a crit. but understated failure mode. To address this, we have designed and fabricated a novel, modified RWV bioreactor capable of continuously removing bubbles while providing optimal fluid dynamics. We validated the capacity of this device with computational and empirical studies. We anticipate that our novel bioreactor will be more consistent and easier to use and may fill a unique and unmet niche in the burgeoning field of organoids.
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  33. 33
    Massai, D.; Isu, G.; Madeddu, D.; Cerino, G.; Falco, A.; Frati, C.; Gallo, D.; Deriu, M. A.; Falvo D’Urso Labate, G.; Quaini, F.; Audenino, A.; Morbiducci, U. A Versatile Bioreactor for Dynamic Suspension Cell Culture. Application to the Culture of Cancer Cell Spheroids. PLoS One 2016, 11 (5), e0154610,  DOI: 10.1371/journal.pone.0154610
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    A versatile bioreactor for dynamic suspension cell culture. Application to the culture of cancer cell spheroids
    Massai, Diana; Isu, Giuseppe; Madeddu, Denise; Cerino, Giulia; Falco, Angela; Frati, Caterina; Gallo, Diego; Deriu, Marco A.; Labate, Giuseppe Falvo D'Urso; Quaini, Federico; Audenino, Alberto; Morbiducci, Umberto
    PLoS One (2016), 11 (5), e0154610/1-e0154610/16CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
    A versatile bioreactor suitable for dynamic suspension cell culture under tunable shear stress conditions has been developed and preliminarily tested culturing cancer cell spheroids. By adopting simple technol. solns. and avoiding rotating components, the bioreactor exploits the laminar hydrodynamics establishing within the culture chamber enabling dynamic cell suspension in an environment favorable to mass transport, under a wide range of tunable shear stress conditions. The design phase of the device has been supported by multiphysics modeling and has provided a comprehensive anal. of the operating principles of the bioreactor. Moreover, an explanatory example is herein presented with multiphysics simulations used to set the proper bioreactor operating conditions for preliminary in vitro biol. tests on a human lung carcinoma cell line. The biol. results demonstrate that the ultralow shear dynamic suspension provided by the device is beneficial for culturing cancer cell spheroids. In comparison to the static suspension control, dynamic cell suspension preserves morphol. features, promotes intercellular connection, increases spheroid size (2.4-fold increase) and no. of cycling cells (1.58-fold increase), and reduces double strand DNA damage (1.5-fold redn.). It is envisioned that the versatility of this bioreactor could allow investigation and expansion of different cell types in the future.
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  34. 34
    Raghavan, S.; Mehta, P.; Horst, E. N.; Ward, M. R.; Rowley, K. R.; Mehta, G. Comparative Analysis of Tumor Spheroid Generation Techniques for Differential in Vitro Drug Toxicity. Oncotarget 2016, 7 (13), 16948– 16961,  DOI: 10.18632/oncotarget.7659
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    Comparative analysis of tumor spheroid generation techniques for differential in vitro drug toxicity
    Raghavan Shreya; Mehta Pooja; Ward Maria R; Mehta Geeta; Horst Eric N; Rowley Katelyn R; Mehta Geeta; Mehta Geeta
    Oncotarget (2016), 7 (13), 16948-61 ISSN:.
    Multicellular tumor spheroids are powerful in vitro models to perform preclinical chemosensitivity assays. We compare different methodologies to generate tumor spheroids in terms of resultant spheroid morphology, cellular arrangement and chemosensitivity. We used two cancer cell lines (MCF7 and OVCAR8) to generate spheroids using i) hanging drop array plates; ii) liquid overlay on ultra-low attachment plates; iii) liquid overlay on ultra-low attachment plates with rotating mixing (nutator plates). Analysis of spheroid morphometry indicated that cellular compaction was increased in spheroids generated on nutator and hanging drop array plates. Collagen staining also indicated higher compaction and remodeling in tumor spheroids on nutator and hanging drop arrays compared to conventional liquid overlay. Consequently, spheroids generated on nutator or hanging drop plates had increased chemoresistance to cisplatin treatment (20-60% viability) compared to spheroids on ultra low attachment plates (10-20% viability). Lastly, we used a mathematical model to demonstrate minimal changes in oxygen and cisplatin diffusion within experimentally generated spheroids. Our results demonstrate that in vitro methods of tumor spheroid generation result in varied cellular arrangement and chemosensitivity.
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    Ko, E.; Poon, M. L. S.; Park, E.; Cho, Y.; Shin, J. H. Engineering 3D Cortical Spheroids for an in Vitro Ischemic Stroke Model. ACS Biomater. Sci. Eng. 2021, 7 (8), 3845– 3860,  DOI: 10.1021/acsbiomaterials.1c00406
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    Engineering 3D Cortical Spheroids for an In Vitro Ischemic Stroke Model
    Ko, Eunmin; Poon, Mong Lung Steve; Park, Eunyoung; Cho, Youngbin; Shin, Jennifer H.
    ACS Biomaterials Science & Engineering (2021), 7 (8), 3845-3860CODEN: ABSEBA; ISSN:2373-9878. (American Chemical Society)
    Three-dimensional (3D) spheroids composed of brain cells have shown great potential to mimic the pathophysiol. of the brain. However, a 3D spheroidal brain-disease model for cerebral ischemia has not been reported. This study investigated an ultralow attachment (ULA) surface-mediated formation of 3D cortical spheroids using primary rat cortical cells to recapitulate the cerebral ischemic responses in stroke by oxygen-glucose deprivation-reoxygenation (OGD-R) treatment. Comparison between two-dimensional (2D) and 3D cell culture models confirmed the better performance of the 3D cortical spheroids as normal brain models. The cortical cells cultured in 3D maintained their healthy physiol. morphol. of a less activated state and suppressed mRNA expressions of pathol. stroke markers, S100B, IL-1β, and MBP, selected based on in vivo stroke model. Interestingly, the spheroids formed on the ULA surface exhibited striking aggregation dynamics involving active cell-substrate interactions, whereas those formed on the agarose surface aggregated passively by the convective flow of the media. Accordingly, ULA spheroids manifested a layered arrangement of neurons and astrocytes with higher expressions of integrin β1, integrin α5, N-cadherin, and fibronectin than the agarose spheroids. OGD-R-induced stroke model of the ULA spheroids successfully mimicked the ischemic response as evidenced by the upregulated mRNA expressions of the key markers for stroke, S100B, IL-1β, and MBP. Our study suggested that structurally and functionally distinct cortical spheroids could be generated by simply tuning the cell-substrate binding activities during dynamic spheroidal formation, which should be an essential factor to consider in establishing a brain-disease model.
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  36. 36
    Vinci, M.; Gowan, S.; Boxall, F.; Patterson, L.; Zimmermann, M.; Court, W.; Lomas, C.; Mendiola, M.; Hardisson, D.; Eccles, S. A. Advances in Establishment and Analysis of Three-Dimensional Tumor Spheroid-Based Functional Assays for Target Validation and Drug Evaluation. BMC Biol. 2012, 10 (March).  DOI: 10.1186/1741-7007-10-29 .
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    Shi, W.; Kwon, J.; Huang, Y.; Tan, J.; Uhl, C. G.; He, R.; Zhou, C.; Liu, Y. Facile Tumor Spheroids Formation in Large Quantity with Controllable Size and High Uniformity. Sci. Rep. 2018, 8 (1), 1– 9,  DOI: 10.1038/s41598-018-25203-3
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    Six years of grazing exclusion is the optimum duration in the alpine meadow-steppe of the north-eastern Qinghai-Tibetan Plateau
    Li, Wen; Liu, Yuzhen; Wang, Jinlan; Shi, Shangli; Cao, Wenxia
    Scientific Reports (2018), 8 (1), 1-13CODEN: SRCEC3; ISSN:2045-2322. (Nature Research)
    Grazing exclusion is an effective management strategy for restoring degraded grasslands worldwide, but the effects of different exclusion durations on vegetation structure and soil properties remain unclear. Therefore, we evaluated vegetation characteristics and soil properties in an alpine meadow-steppe under grazing exclusion of different lengths (with grazing and with 3-yr, 6-yr, 9-yr and 11-yr grazing exclusions) on the Qinghai-Tibetan Plateau (QTP). We also explored the relationships among above-ground biomass, biodiversity and soil properties to ascertain the mechanism underlying the impact of grazing exclusion on these factors. The results showed that the above- and below-ground biomass, total no. of plant species, community d., Shannon-Wiener diversity index, evenness index, richness index, soil and vegetation carbon (C) and nitrogen (N) storage and ecosystem C and N storage exhibited a hump-shaped pattern in response to the length of grazing exclusion with a 6-yr threshold. In addn., structural equation modeling showed that the bulk d., soil moisture content, micro sand content and clay and silt contents were the most important detg. factors leading to an increase in above-ground biomass in the alpine meadow-steppe after grazing exclusion, whereas the soil total N, available N, available phosphate and soil org. C content were the most important detg. factors leading to a decrease in biodiversity. Considering the stability of the plant community and the C and N pools, long-term grazing exclusion (>9 years) is unnecessary, and the optimum exclosure duration of the moderately degraded Elymus nutans - Kobresia humilis type alpine meadow-steppe is six years on the north-eastern QTP.
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  38. 38
    Vadivelu, R. K.; Ooi, C. H.; Yao, R. Q.; Tello Velasquez, J.; Pastrana, E.; Diaz-Nido, J.; Lim, F.; Ekberg, J. A. K.; Nguyen, N. T.; St John, J. A. Generation of Three-Dimensional Multiple Spheroid Model of Olfactory Ensheathing Cells Using Floating Liquid Marbles. Sci. Rep. 2015,  DOI: 10.1038/srep15083
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    Li, H.; Liu, P.; Kaur, G.; Yao, X.; Yang, M. Transparent and Gas-Permeable Liquid Marbles for Culturing and Drug Sensitivity Test of Tumor Spheroids. Adv. Healthc. Mater. 2017, 6, 1700185,  DOI: 10.1002/adhm.201700185
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    Chen, M.; Shah, M. P.; Shelper, T. B.; Nazareth, L.; Barker, M.; Tello Velasquez, J.; Ekberg, J. A. K.; Vial, M. L.; St John, J. A. Naked Liquid Marbles: A Robust Three-Dimensional Low-Volume Cell-Culturing System. ACS Appl. Mater. Interfaces 2019, 11, 9814,  DOI: 10.1021/acsami.8b22036
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    Naked Liquid Marbles: A Robust Three-Dimensional Low-Volume Cell-Culturing System
    Chen, Mo; Shah, Megha P.; Shelper, Todd B.; Nazareth, Lynn; Barker, Matthew; Tello Velasquez, Johana; Ekberg, Jenny A. K.; Vial, Marie-Laure; St. John, James A.
    ACS Applied Materials & Interfaces (2019), 11 (10), 9814-9823CODEN: AAMICK; ISSN:1944-8244. (American Chemical Society)
    Three-dimensional (3D) multicellular structures allow cells to behave and interact with each other in a manner that mimics the in vivo environment. In recent years, many 3D cell culture methods have been developed with the goal of producing the most in vivo-like structures possible. While strongly preferable to conventional cell culture, these approaches are often poorly reproducible, time-consuming, expensive, and labor-intensive and require specialized equipment. Here, the authors describe a novel 3D culture platform, which the authors have termed the naked liq. marble (NLM). Cells are cultured in a liq. drop (the NLM) in superhydrophobic-coated plates, which causes the cells to naturally form 3D structures. Inside the NLMs, cells are free to interact with each other, forming multiple 3D spheroids that are uniform in size and shape in less than 24 h. The authors showed that this system is highly reproducible, suitable for cell coculture, compd. screening, and also compatible with lab. automation systems. The low cost of prodn., small vol. of each NLM, and prodn. via automated liq. handling make this 3D cell-culturing system particularly suitable for high-throughput screening assays such as drug testing as well as numerous other cell-based research applications.
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    Oliveira, N. M.; Correia, C. R.; Reis, R. L.; Mano, J. F. Liquid Marbles for High-Throughput Biological Screening of Anchorage-Dependent Cells. Adv. Healthc. Mater. 2015, 4, 264,  DOI: 10.1002/adhm.201400310
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    41
    Liquid Marbles for High-Throughput Biological Screening of Anchorage-Dependent Cells
    Oliveira, Nuno M.; Correia, Clara R.; Reis, Rui L.; Mano, Joao F.
    Advanced Healthcare Materials (2015), 4 (2), 264-270CODEN: AHMDBJ; ISSN:2192-2640. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Stable liq. marbles (LM) are produced by coating liq. droplets with a hydrophobic powder. The used hydrophobic powder is produced by fluorosi-lanization of diatomaceous earth, used before to produce superhydrophobic structures. Here, the use of LM is proposed for high-throughput drug screening on anchorage-dependent cells. To provide the required cell adhesion sites inside the liq. environment of LM, surface-modified poly(l-lactic acid) microparticles are used. A simple method that takes advantage from LM appealing features is presented, such as the ability to inject liq. on LM without disrupting (self-healing ability), and to monitor color changes inside of LM. After promoting cell adhesion, a cytotoxic screening test is performed as a proof of concept. Fe3+ is used as a model cytotoxic agent and is injected on LM. After incubation, AlamarBlue reagent is injected and used to assess the presence of viable cells, by monitoring color change from blue to red. Color intensity is measured by image processing and the anal. of pictures takes using an ordinary digital camera. The proposed method is fully validated in counterpoint to an MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) colorimetric assay, a well-known method used for the cytotoxicity assessment.
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    Yaman, S.; Anil-Inevi, M.; Ozcivici, E.; Tekin, H. C. Magnetic Force-Based Microfluidic Techniques for Cellular and Tissue Bioengineering. Frontiers in Bioengineering and Biotechnology. 2018,  DOI: 10.3389/fbioe.2018.00192
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    Anil-Inevi, M.; Yaman, S.; Yildiz, A. A.; Mese, G.; Yalcin-Ozuysal, O.; Tekin, H. C.; Ozcivici, E. Biofabrication of in Situ Self Assembled 3D Cell Cultures in a Weightlessness Environment Generated Using Magnetic Levitation. Sci. Rep. 2018,  DOI: 10.1038/s41598-018-25718-9
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    Durmus, N. G.; Tekin, H. C.; Guven, S.; Sridhar, K.; Arslan Yildiz, A.; Calibasi, G.; Ghiran, I.; Davis, R. W.; Steinmetz, L. M.; Demirci, U. Magnetic Levitation of Single Cells. Proc. Natl. Acad. Sci. U. S. A. 2015, 112, E3661– E3668,  DOI: 10.1073/pnas.1509250112
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    Magnetic levitation of single cells
    Durmus, Naside Gozde; Tekin, H. Cumhur; Guven, Sinan; Sridhar, Kaushik; Yildiz, Ahu Arslan; Calibasi, Gizem; Ghiran, Ionita; Davis, Ronald W.; Steinmetz, Lars M.; Demirci, Utkan
    Proceedings of the National Academy of Sciences of the United States of America (2015), 112 (28), E3661-E3668CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Several cellular events cause permanent or transient changes in inherent magnetic and d. properties of cells. Characterizing these changes in cell populations is crucial to understand cellular heterogeneity in cancer, immune response, infectious diseases, drug resistance, and evolution. Although magnetic levitation has previously been used for macroscale objects, its use in life sciences has been hindered by the inability to levitate microscale objects and by the toxicity of metal salts previously applied for levitation. Here, the authors use magnetic levitation principles for biol. characterization and monitoring of cells and cellular events. Each cell type (i.e., cancer, blood, bacteria, and yeast) has a characteristic levitation profile, which the authors distinguish at an unprecedented resoln. of 1 × 10-4 g·mL-1. The authors have identified unique differences in levitation and d. blueprints between breast, esophageal, colorectal, and nonsmall cell lung cancer cell lines, as well as heterogeneity within these seemingly homogeneous cell populations. Furthermore, changes in cellular d. and levitation profiles can be monitored in real time at single-cell resoln., allowing quantification of heterogeneous temporal responses of each cell to environmental stressors. These data establish d. as a powerful biomarker for studying living systems and their responses. Thereby, the authors' method enables rapid, d.-based imaging and profiling of single cells with intriguing applications, such as label-free identification and monitoring of heterogeneous biol. changes under various physiol. conditions, including antibiotic or cancer treatment in personalized medicine.
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    Anil-Inevi, M.; Delikoyun, K.; Mese, G.; Tekin, H. C.; Ozcivici, E. Magnetic Levitation Assisted Biofabrication, Culture, and Manipulation of 3D Cellular Structures Using a Ring Magnet Based Setup. Biotechnol. Bioeng. 2021, 118, 4771,  DOI: 10.1002/bit.27941
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    Magnetic levitation assisted biofabrication, culture, and manipulation of 3D cellular structures using a ring magnet based setup
    Anil-Inevi, Muge; Delikoyun, Kerem; Mese, Gulistan; Tekin, H. Cumhur; Ozcivici, Engin
    Biotechnology and Bioengineering (2021), 118 (12), 4771-4785CODEN: BIBIAU; ISSN:0006-3592. (John Wiley & Sons, Inc.)
    Diamagnetic levitation is an emerging technol. for remote manipulation of cells in cell and tissue level applications. Low-cost magnetic levitation configurations using permanent magnets are commonly composed of a culture chamber phys. sandwiched between two block magnets that limit working vol. and applicability. This work describes a single ring magnet-based magnetic levitation system to eliminate phys. limitations for biofabrication. Developed configuration utilizes sample culture vol. for construct size manipulation and long-term maintenance. Furthermore, our configuration enables convenient transfer of liq. or solid phases during the levitation. Before biofabrication, we first calibrated/ the platform for levitation with polymeric beads, considering the single cell d. range of viable cells. By taking advantage of magnetic focusing and cellular self-assembly, millimeter-sized 3D structures were formed and maintained in the system allowing easy and on-site intervention in cell culture with an open operational space. We demonstrated that the levitation protocol could be adapted for levitation of various cell types (i.e., stem cell, adipocyte and cancer cell) representing cells of different densities by modifying the paramagnetic ion concn. that could be also reduced by manipulating the d. of the medium. This technique allowed the manipulation and merging of sep. formed 3D biol. units, as well as the hybrid biofabrication with biopolymers. In conclusion, we believe that this platform will serve as an important tool in broad fields such as bottom-up tissue engineering, drug discovery and developmental biol.
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  46. 46
    Sarigil, O.; Anil-Inevi, M.; Firatligil-Yildirir, B.; Unal, Y. C.; Yalcin-Ozuysal, O.; Mese, G.; Tekin, H. C.; Ozcivici, E. Scaffold-Free Biofabrication of Adipocyte Structures with Magnetic Levitation. Biotechnol. Bioeng. 2021, 118, 1127,  DOI: 10.1002/bit.27631
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    Scaffold-free biofabrication of adipocyte structures with magnetic levitation
    Sarigil, Oyku; Anil-Inevi, Muge; Firatligil-Yildirir, Burcu; Unal, Yagmur Ceren; Yalcin-Ozuysal, Ozden; Mese, Gulistan; Tekin, H. Cumhur; Ozcivici, Engin
    Biotechnology and Bioengineering (2021), 118 (3), 1127-1140CODEN: BIBIAU; ISSN:0006-3592. (John Wiley & Sons, Inc.)
    Tissue engineering research aims to repair the form and/or function of impaired tissues. Tissue engineering studies mostly rely on scaffold-based techniques. However, these techniques have certain challenges, such as the selection of proper scaffold material, including mech. properties, sterilization, and fabrication processes. As an alternative, we propose a novel scaffold-free adipose tissue biofabrication technique based on magnetic levitation. In this study, a label-free magnetic levitation technique was used to form three-dimensional scaffold-free adipocyte structures with various fabrication strategies in a microcapillary-based setup. Adipogenic-differentiated 7F2 cells and growth D1 ORL UVA stem cells were used as model cells. The morphol. properties of the 3D structures of single and cocultured cells were analyzed. The developed procedure leads to the formation of different patterns of single and cocultured adipocytes without a scaffold. Our results indicated that adipocytes formed loose structures while growth cells were tightly packed during 3D culture in the magnetic levitation platform. This system has potential for ex vivo modeling of adipose tissue for drug testing and transplantation applications for cell therapy in soft tissue damage. Also, it will be possible to extend this technique to other cell and tissue types.
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    Ino, K.; Ito, A.; Honda, H. Cell Patterning Using Magnetite Nanoparticles and Magnetic Force. Biotechnol. Bioeng. 2007, 97, 1309,  DOI: 10.1002/bit.21322
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    Cell patterning using magnetite nanoparticles and magnetic force
    Ino, Kosuke; Ito, Akira; Honda, Hiroyuki
    Biotechnology and Bioengineering (2007), 97 (5), 1309-1317CODEN: BIBIAU; ISSN:0006-3592. (John Wiley & Sons, Inc.)
    Technologies for fabricating functional tissue architectures by patterning cells precisely are highly desirable for tissue engineering. Although several cell patterning methods such as microcontact printing and lithog. have been developed, these methods require specialized surfaces to be used as substrates, the fabrication of which is time consuming. In the present study, we demonstrated a simple and rapid cell patterning technique, using magnetite nanoparticles and magnetic force, which enables us to allocate cells on arbitrary surfaces. Magnetite cationic liposomes (MCLs) developed in our previous study were used to magnetically label the target cells. When steel plates placed on a magnet were positioned under a cell culture surface, the magnetically labeled cells lined on the surface where the steel plate was positioned. Patterned lines of single cells were achieved by adjusting the no. of cells seeded, and complex cell patterns (curved, parallel, or crossing patterns) were successfully fabricated. Since cell patterning using magnetic force may not limit the property of culture surfaces, human umbilical vein endothelial cells (HUVECs) were patterned on Matrigel, thereby forming patterned capillaries. These results suggest that the novel cell patterning methodol., which uses MCLs, is a promising approach for tissue engineering and studying cell-cell interactions in vitro.
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    Li, Y.; Kumacheva, E. Hydrogel Microenvironments for Cancer Spheroid Growth and Drug Screening. Science Advances. 2018,  DOI: 10.1126/sciadv.aas8998
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    Costa, E. C.; Moreira, A. F.; de Melo-Diogo, D.; Gaspar, V. M.; Carvalho, M. P.; Correia, I. J. 3D Tumor Spheroids: An Overview on the Tools and Techniques Used for Their Analysis. Biotechnology Advances. 2016, 34, 1427,  DOI: 10.1016/j.biotechadv.2016.11.002
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    3D tumor spheroids: an overview on the tools and techniques used for their analysis
    Costa Elisabete C; Moreira Andre F; de Melo-Diogo Duarte; Gaspar Vitor M; Carvalho Marco P; Correia Ilidio J
    Biotechnology advances (2016), 34 (8), 1427-1441 ISSN:.
    In comparison with 2D cell culture models, 3D spheroids are able to accurately mimic some features of solid tumors, such as their spatial architecture, physiological responses, secretion of soluble mediators, gene expression patterns and drug resistance mechanisms. These unique characteristics highlight the potential of 3D cellular aggregates to be used as in vitro models for screening new anticancer therapeutics, both at a small and large scale. Nevertheless, few reports have focused on describing the tools and techniques currently available to extract significant biological data from these models. Such information will be fundamental to drug and therapeutic discovery process using 3D cell culture models. The present review provides an overview of the techniques that can be employed to characterize and evaluate the efficacy of anticancer therapeutics in 3D tumor spheroids.
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    Kim, M.; Mun, H.; Sung, C. O.; Cho, E. J.; Jeon, H. J.; Chun, S. M.; Jung, D. J.; Shin, T. H.; Jeong, G. S.; Kim, D. K.; Choi, E. K.; Jeong, S. Y.; Taylor, A. M.; Jain, S.; Meyerson, M.; Jang, S. J. Patient-Derived Lung Cancer Organoids as in Vitro Cancer Models for Therapeutic Screening. Nat. Commun. 2019,  DOI: 10.1038/s41467-019-11867-6
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    Ruedinger, F.; Lavrentieva, A.; Blume, C.; Pepelanova, I.; Scheper, T. Hydrogels for 3D Mammalian Cell Culture: A Starting Guide for Laboratory Practice. Appl. Microbiol. Biotechnol. 2015, 99, 623,  DOI: 10.1007/s00253-014-6253-y
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    Hydrogels for 3D mammalian cell culture: a starting guide for laboratory practice
    Ruedinger, Ferdinand; Lavrentieva, Antonina; Blume, Cornelia; Pepelanova, Iliyana; Scheper, Thomas
    Applied Microbiology and Biotechnology (2015), 99 (2), 623-636CODEN: AMBIDG; ISSN:0175-7598. (Springer)
    A review. Hydrogels have become one of the most popular platforms for three-dimensional (3D) cultivation of mammalian cells. The enormous versatility of hydrogel materials makes it possible to design scaffolds with predefined mech. properties, as well as with desired biofunctionality. 3D hydrogel constructs have been used for a variety of applications, including tissue engineering of microorgan systems, drug delivery, cytotoxicity testing, and drug screening. Moreover, 3D culture is applied for investigating cellular physiol., stem cell differentiation, and tumor models and for studying interaction mechanisms between the extracellular matrix and cells. In this paper, we review current examples of performance-based hydrogel design for 3D cell culture applications. A major emphasis is placed on a description of how std. anal. protocols and imaging techniques are being adapted to anal. of 3D cell culture in hydrogel systems.
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    Sauty, B.; Santesarti, G.; Fleischhammer, T.; Lindner, P.; Lavrentieva, A.; Pepelanova, I.; Marino, M. Enabling Technologies for Obtaining Desired Stiffness Gradients in GelMA Hydrogels Constructs. Macromol. Chem. Phys. 2022, 223 (2), 2100326,  DOI: 10.1002/macp.202100326
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    Enabling Technologies for Obtaining Desired Stiffness Gradients in GelMA Hydrogels Constructs
    Sauty, Bastien; Santesarti, Gianluca; Fleischhammer, Tabea; Lindner, Patrick; Lavrentieva, Antonina; Pepelanova, Iliyana; Marino, Michele
    Macromolecular Chemistry and Physics (2022), 223 (2), 2100326CODEN: MCHPES; ISSN:1022-1352. (Wiley-VCH Verlag GmbH & Co. KGaA)
    This work presents enabling technologies for the optimization of the manufg. of GelMA-based hydrogels constructs with desired stiffness gradients. The manufg. technique combines dynamic mixing for gradient generation and a passive micromixer for efficient hydrogel blending. A digital replica of the fabrication process is developed, integrating theor. and computational models, as well as exptl. data, in order to predict and control the stiffness profile obtained within the constructs. The workflow for the development of the in silico framework, based on rigorous verification, validation, and uncertainty quantification steps, is presented. The validation of the digital replica is based on ref. settings of process variables, which result in constructs with an exponential stiffness profile. The developed in silico model has been employed for optimizing process variables in order to obtain a linear stiffness profile in the extruded construct without the need of expensive and time-consuming trial-and-error procedures. The developed digital replica is now a powerful tool for the creation of hydrogel gradient constructs for tissue engineering applications or for the screening of optimal 3D cell culture conditions.
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    van Duinen, V.; Trietsch, S. J.; Joore, J.; Vulto, P.; Hankemeier, T. Microfluidic 3D Cell Culture: From Tools to Tissue Models. Current Opinion in Biotechnology. 2015, 35, 118,  DOI: 10.1016/j.copbio.2015.05.002
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    Microfluidic 3D cell culture: from tools to tissue models
    van Duinen, Vincent; Trietsch, Sebastiaan J.; Joore, Jos; Vulto, Paul; Hankemeier, Thomas
    Current Opinion in Biotechnology (2015), 35 (), 118-126CODEN: CUOBE3; ISSN:0958-1669. (Elsevier B.V.)
    A review. The transition from 2D to 3D cell culture techniques is an important step in a trend towards better biomimetic tissue models. Microfluidics that allows spatial control over fluids in micrometer-sized channels has become a valuable tool to further increase the physiol. relevance of 3D cell culture by enabling spatially controlled co-cultures, perfusion flow and spatial control over signaling gradients. This paper reviews the most important developments in microfluidic 3D culture since 2012. Most efforts were exerted in the field of vasculature, both as a tissue on its own and as part of cancer models. The focus is shifting from tool building to implementation of specific tissue models. The next big challenge for the field is the full validation of these models and subsequently the implementation of these models in drug development pipelines of the pharmaceutical industry and ultimately in personalized medicine applications.
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    Wu, X.; Su, J.; Wei, J.; Jiang, N.; Ge, X. Recent Advances in Three-Dimensional Stem Cell Culture Systems and Applications. Stem Cells International. 2021, 2021, 1,  DOI: 10.1155/2021/9477332
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    Inamdar, N. K.; Borenstein, J. T. Microfluidic Cell Culture Models for Tissue Engineering. Current Opinion in Biotechnology. 2011, 22, 681,  DOI: 10.1016/j.copbio.2011.05.512
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    Microfluidic cell culture models for tissue engineering
    Inamdar, Niraj K.; Borenstein, Jeffrey T.
    Current Opinion in Biotechnology (2011), 22 (5), 681-689CODEN: CUOBE3; ISSN:0958-1669. (Elsevier B.V.)
    A review. Microfluidic systems have emerged as revolutionary new platform technologies for a range of applications, from consumer products such as inkjet printer cartridges to lab-on-a-chip diagnostic systems. Recent developments have opened the door to a new set of opportunities for microfluidic systems, in the field of tissue and organ engineering. Advances in the design of physiol. relevant structures and networks, fabrication processes for biomaterials suitable for in vivo use, and techniques for scaling towards large, 3-dimensional constructs, are converging towards therapeutic applications of microfluidic technologies in engineering complex tissues and organs. These advances herald a new generation of microfluidics-based approaches designed for specific tissue and organ applications, incorporating microvascular networks, structures for transport and filtration, and a 3-dimensional microenvironment suitable for supporting phenotypic cell behavior, tissue function, and implantation and host integration.
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    Harink, B.; Le Gac, S.; Truckenmüller, R.; Van Blitterswijk, C.; Habibovic, P. Regeneration-on-a-Chip? The Perspectives on Use of Microfluidics in Regenerative Medicine. Lab on a Chip. 2013, 13, 3512,  DOI: 10.1039/c3lc50293g
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    Regeneration-on-a-chip? The perspectives on use of microfluidics in regenerative medicine
    Harink, Bjoern; Le Gac, Severine; Truckenmueller, Roman; van Blitterswijk, Clemens; Habibovic, Pamela
    Lab on a Chip (2013), 13 (18), 3512-3528CODEN: LCAHAM; ISSN:1473-0189. (Royal Society of Chemistry)
    A review. The aim of regenerative medicine is to restore or establish normal function of damaged tissues or organs. Tremendous efforts are placed into development of novel regenerative strategies, involving (stem) cells, sol. factors, biomaterials or combinations thereof, as a result of the growing need caused by continuous population aging. To satisfy this need, fast and reliable assessment of (biol.) performance is sought, not only to select the potentially interesting candidates, but also to rule out poor ones at an early stage of development. Microfluidics may provide a new avenue to accelerate research and development in the field of regenerative medicine as it has proven its maturity for the realization of high-throughput screening platforms. In addn., microfluidic systems offer other advantages such as the possibility to create in vivo-like microenvironments. Besides the complexity of organs or tissues that need to be regenerated, regenerative medicine brings addnl. challenges of complex regeneration processes and strategies. The question therefore arises whether so much complexity can be integrated into microfluidic systems without compromising reliability and throughput of assays. With this review, the authors aim to investigate whether microfluidics can become widely applied in regenerative medicine research and/or strategies.
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  57. 57
    Damiati, S.; Kompella, U. B.; Damiati, S. A.; Kodzius, R. Microfluidic Devices for Drug Delivery Systems and Drug Screening. Genes. 2018, 9, 103,  DOI: 10.3390/genes9020103
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    57
    Microfluidic devices for drug delivery systems and drug screening
    Damiati, Samar; Kompella, Uday B.; Damiati, Safa A.; Kodzius, Rimantas
    Genes (2018), 9 (2), 103/1-103/24CODEN: GENEG9; ISSN:2073-4425. (MDPI AG)
    Microfluidic devices present unique advantages for the development of efficient drug carrier particles, cell-free protein synthesis systems, and rapid techniques for direct drug screening. Compared to bulk methods, by efficiently controlling the geometries of the fabricated chip and the flow rates of multiphase fluids, microfluidic technol. enables the generation of highly stable, uniform, monodispersed particles with higher encapsulation efficiency. Since the existing preclin. models are inefficient drug screens for predicting clin. outcomes, microfluidic platforms might offer a more rapid and cost-effective alternative. Compared to 2D cell culture systems and in vivo animal models, microfluidic 3D platforms mimic the in vivo cell systems in a simple, inexpensive manner, which allows high throughput and multiplexed drug screening at the cell, organ, and whole-body levels. In this review, the generation of appropriate drug or gene carriers including different particle types using different configurations of microfluidic devices is highlighted. Addnl., this paper discusses the emergence of fabricated microfluidic cell-free protein synthesis systems for potential use at point of care as well as cell-, organ-, and human-on-a-chip models as smart, sensitive, and reproducible platforms, allowing the investigation of the effects of drugs under conditions imitating the biol. system.
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  58. 58
    Li, Y.; Li, D.; Zhao, P.; Nandakumar, K.; Wang, L.; Song, Y. Microfluidics-Based Systems in Diagnosis of Alzheimer’s Disease and Biomimetic Modeling. Micromachines. 2020, 11, 787,  DOI: 10.3390/mi11090787
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    Lee, S. A.; No, D. Y.; Kang, E.; Ju, J.; Kim, D. S.; Lee, S. H. Spheroid-Based Three-Dimensional Liver-on-a-Chip to Investigate Hepatocyte-Hepatic Stellate Cell Interactions and Flow Effects. Lab Chip 2013, 13, 3529,  DOI: 10.1039/c3lc50197c
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    59
    Spheroid-based three-dimensional liver-on-a-chip to investigate hepatocyte-hepatic stellate cell interactions and flow effects
    Lee, Seung-A.; No, Da Yoon; Kang, Edward; Ju, Jongil; Kim, Dong-Sik; Lee, Sang-Hoon
    Lab on a Chip (2013), 13 (18), 3529-3537CODEN: LCAHAM; ISSN:1473-0189. (Royal Society of Chemistry)
    We have developed a three-dimensional (3D) liver-on-a-chip to investigate the interaction of hepatocytes and hepatic stellate cells (HSCs) in which primary 3D hepatocyte spheroids and HSCs are co-cultured without direct cell-cell contact. Here, we show that the 3D liver chip offers substantial advantages for the formation and harvesting of spheroids. The most important feature of this liver chip is that it enables continuous flow of medium to the cells through osmotic pumping, and thus requires only minimal handling and no external power source. We also demonstrate that flow assists the formation and long-term maintenance of spheroids. Addnl., we quant. and qual. investigated the paracrine effects of HSCs, demonstrating that HSCs assist in the maintenance of hepatocyte spheroids and play an important role in the formation of tight cell-cell contacts, thereby improving liver-specific function. Spheroids derived from co-cultures exhibited improved albumin and urea secretion rates compared to mono-cultured spheroids after 9 days. Immunostaining for cytochrome P 450 revealed that the enzymic activity of spheroids co-cultured for 8 days was greater than that of mono-cultured spheroids. These results indicate that this system has the potential for further development as a unique model for studying cellular interactions or as a tool that can be incorporated into other models aimed at creating hepatic structure and prolonging hepatocyte function in culture.
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  60. 60
    Li, X.; Valadez, A. V.; Zuo, P.; Nie, Z. Microfluidic 3D Cell Culture: Potential Application for Tissue-Based Bioassays. Bioanalysis. 2012, 4, 1509,  DOI: 10.4155/bio.12.133
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    Microfluidic 3D cell culture: potential application for tissue-based bioassays
    Li, XiuJun; Valadez, Alejandra V.; Zuo, Peng; Nie, Zhihong
    Bioanalysis (2012), 4 (12), 1509-1525CODEN: BIOAB4; ISSN:1757-6180. (Future Science Ltd.)
    Review. Current fundamental investigations of human biol. and the development of therapeutic drugs commonly rely on 2D monolayer cell culture systems. However, 2D cell culture systems do not accurately recapitulate the structure, function or physiol. of living tissues, nor the highly complex and dynamic 3D environments in vivo. Microfluidic technol. can provide microscale complex structures and well-controlled parameters to mimic the in vivo environment of cells. The combination of microfluidic technol. with 3D cell culture offers great potential for in vivo-like tissue-based applications, such as the emerging organ-on-a-chip system. This article will review recent advances in the microfluidic technol. for 3D cell culture and their biol. applications.
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    Sart, S.; Tomasi, R. F. X.; Amselem, G.; Baroud, C. N. Multiscale Cytometry and Regulation of 3D Cell Cultures on a Chip. Nat. Commun. 2017,  DOI: 10.1038/s41467-017-00475-x
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    Alessandri, K.; Sarangi, B. R.; Gurchenkov, V. V.; Sinha, B.; Kießling, T. R.; Fetler, L.; Rico, F.; Scheuring, S.; Lamaze, C.; Simon, A.; Geraldo, S.; Vignjević, D.; Doméjean, H.; Rolland, L.; Funfak, A.; Bibette, J.; Bremond, N.; Nassoy, P. Cellular Capsules as a Tool for Multicellular Spheroid Production and for Investigating the Mechanics of Tumor Progression in Vitro. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 14843,  DOI: 10.1073/pnas.1309482110
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    62
    Cellular capsules as a tool for multicellular spheroid production and for investigating the mechanics of tumor progression in vitro
    Alessandri, Kevin; Sarangi, Bibhu Ranjan; Gurchenkov, Vasily Valerievitch; Sinha, Bidisha; Kiessling, Tobias Reinhold; Fetler, Luc; Rico, Felix; Scheuring, Simon; Lamaze, Christophe; Simon, Anthony; Geraldo, Sara; Vignjevic, Danijela; Domejean, Hugo; Rolland, Leslie; Funfak, Anette; Bibette, Jerome; Bremond, Nicolas; Nassoy, Pierre
    Proceedings of the National Academy of Sciences of the United States of America (2013), 110 (37), 14843-14848,S14843/1-S14843/16CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Deciphering the multifactorial determinants of tumor progression requires standardized high-throughput prepn. of 3D in vitro cellular assays. The authors present a simple microfluidic method based on the encapsulation and growth of cells inside permeable, elastic, hollow microspheres. This approach enables mass prodn. of size-controlled multicellular spheroids. Due to their geometry and elasticity, these microcapsules can uniquely serve as quant. mech. sensors to measure the pressure exerted by the expanding spheroid. By monitoring the growth of individual encapsulated spheroids after confluence, the authors dissect the dynamics of pressure buildup toward a steady-state value, consistent with the concept of homeostatic pressure. In turn, these confining conditions increase the cellular d. and affect the cellular organization of the spheroid. Postconfluent spheroids exhibit a necrotic core cemented by a blend of extracellular material and surrounded by a rim of proliferating hypermotile cells. By performing invasion assays in a collagen matrix, the authors report that peripheral cells readily escape preconfined spheroids and cell-cell cohesivity is maintained for freely growing spheroids, suggesting that mech. cues from the surrounding microenvironment may trigger cell invasion from a growing tumor. Overall, the authors' technol. offers a unique avenue to produce in vitro cell-based assays useful for developing new anticancer therapies and to study the interplay between mechanics and growth in tumor evolution.
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    Wang, Y.; Wang, J. Mixed Hydrogel Bead-Based Tumor Spheroid Formation and Anticancer Drug Testing. Analyst 2014, 139, 2449,  DOI: 10.1039/C4AN00015C
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    Mixed hydrogel bead-based tumor spheroid formation and anticancer drug testing
    Wang, Yaolei; Wang, Jinyi
    Analyst (Cambridge, United Kingdom) (2014), 139 (10), 2449-2458CODEN: ANALAO; ISSN:0003-2654. (Royal Society of Chemistry)
    Three-dimensional multicellular tumor spheroids have become crit. for anticancer study since they may provide a better model than conventional monolayer cultures of cancer cells. Various methods for tumor spheroid formation have been explored. However, only one kind of hydrogel was used in these methods, which has an influence on the size and morphol. of the obtained tumor spheroids. Herein, we present a microfluidic droplet-based method for the formation of multicellular tumor spheroids using alginate and matrigel mixed hydrogel beads. By on-chip changing the flow rate of the two hydrogel solns., mixed hydrogel beads with different vol. ratios between alginate and matrigel are obtained. Meanwhile, human cervical carcinoma (HeLa) cells are encapsulated in the mixed hydrogel beads. Acridine orange and propidium iodide double-staining assay shows that the viability of cells encapsulated in the mixed hydrogel beads was more than 90%. After 4 day culture, the multicellular tumor spheroids were successfully formed with spherical shape and uniform size distribution compared with spheroids formed in pure alginate beads. Cytoskeletal anal. by TRITC-phalloidin staining show that HeLa cells in the mixed hydrogel beads closely link to each other. The dose-dependent response assay of HeLa cell spheroids to vincristine show that multicellular spheroids have more powerful resistance to vincristine compared to conventional monolayer culture cells. Taken together, this novel technol. may be of importance to facilitate in vitro culture of tumor spheroids for their ever-increasing utilization in modern cell-based medicine.
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  64. 64
    Chan, H. F.; Zhang, Y.; Ho, Y. P.; Chiu, Y. L.; Jung, Y.; Leong, K. W. Rapid Formation of Multicellular Spheroids in Double-Emulsion Droplets with Controllable Microenvironment. Sci. Rep. 2013,  DOI: 10.1038/srep03462
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    Sabhachandani, P.; Sarkar, S.; Mckenney, S.; Ravi, D.; Evens, A. M.; Konry, T. Microfluidic Assembly of Hydrogel-Based Immunogenic Tumor Spheroids for Evaluation of Anticancer Therapies and Biomarker Release. J. Controlled Release 2019, 295, 21,  DOI: 10.1016/j.jconrel.2018.12.010
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    65
    Microfluidic assembly of hydrogel-based immunogenic tumor spheroids for evaluation of anticancer therapies and biomarker release
    Sabhachandani, Pooja; Sarkar, Saheli; Mckenney, Seamus; Ravi, Dashnamoorthy; Evens, Andrew M.; Konry, Tania
    Journal of Controlled Release (2019), 295 (), 21-30CODEN: JCREEC; ISSN:0168-3659. (Elsevier B.V.)
    Diffuse large B cell lymphoma (DLBCL), the most common subtype of Non-Hodgkin lymphoma, exhibits pathol. heterogeneity and a dynamic immunogenic tumor microenvironment (TME). However, the lack of preclin. in vitro models of DLBCL TME hinders optimal therapeutic screening. This study describes the development of an integrated droplet microfluidics-based platform for high-throughput generation of immunogenic DLBCL spheroids. The spheroids consist of three cell types (cancer, fibroblast and lymphocytes) in a novel hydrogel combination of alginate and puramatrix, which promoted cell adhesion and aggregation. This system facilitates dynamic anal. of cellular interaction, proliferation and therapeutic efficacy via spatiotemporal monitoring and secretome profiling. The immunomodulatory drug lenalidomide had direct anti-proliferative effect on activated B-cell like DLBCL spheroids and reduced several cytokines and other markers (e.g., CCL2, CCL3, CCL4, CD137 and ANG-1 levels) compared with untreated spheroids. Collectively, this novel spheroid platform will enable high-throughput anti-cancer therapeutic screening in a semi-automated manner.
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  66. 66
    Kim, C.; Chung, S.; Kim, Y. E.; Lee, K. S.; Lee, S. H.; Oh, K. W.; Kang, J. Y. Generation of Core-Shell Microcapsules with Three-Dimensional Focusing Device for Efficient Formation of Cell Spheroid. Lab Chip 2011, 11, 246,  DOI: 10.1039/C0LC00036A
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    66
    Generation of core-shell microcapsules with three-dimensional focusing device for efficient formation of cell spheroid
    Kim, Choong; Chung, Seok; Kim, Young Eun; Lee, Kang Sun; Lee, Soo Hyun; Oh, Kwang Wook; Kang, Ji Yoon
    Lab on a Chip (2011), 11 (2), 246-252CODEN: LCAHAM; ISSN:1473-0197. (Royal Society of Chemistry)
    The authors present a microfluidic device generating three-dimensional (3D) coaxial flow by the addn. of a simple hillock to produce an alginate core-shell microcapsule for the efficient formation of a cell spheroid. A hillock tapered at downstream of the two-dimensional focusing channel enables outside flow to enclose the core flow. The aq. soln. in the core flow was focused and surrounded by 1.8% alginate soln. to be solidified as a shell. The double-layered coaxial flow (aq. phase) was broken up into a droplet by the shear flow of oleic acid (oil phase) contg. calcium chloride for the polymn. of the alginate shell. The droplet generated from the laminar coaxial flow maintained a double-layer structure and gelation of the alginate soln. made a core-shell microcapsule. The shell-thickness of the microcapsule was adjusted from 8-21 μm by the variation of two aq. flow rates. The inner shape of the shell was almost spherical when the ratio of the water-glycol mixt. in the core flow exceeded 20%. The microcapsule was used to form a spheroid of embryonic carcinoma cells (embryoid body; EB) by injecting a cell suspension into the core flow. The cells inside the microcapsule aggregated into an EB within 2 days and the EB formation rate was more than 80% with strong compaction. The microcapsule formed single spherical EBs without small satellite clusters or a bumpy shape as obsd. in solid microbeads. The microfluidic chip for encapsulation of cells could generate a no. of EBs with high rate of EB formation when compared with the conventional hanging drop method. The core-shell microcapsule generated by 3D focusing in the microchannel was effective in forming large no. of spherical cell clusters and the encapsulation of cells in the microcapsule is expected to be useful in the transplantation of islet cells or cancer stem cell enrichment.
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    Sun, Q.; Tan, S. H.; Chen, Q.; Ran, R.; Hui, Y.; Chen, D.; Zhao, C. X. Microfluidic Formation of Coculture Tumor Spheroids with Stromal Cells As a Novel 3D Tumor Model for Drug Testing. ACS Biomater. Sci. Eng. 2018, 4, 4425,  DOI: 10.1021/acsbiomaterials.8b00904
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    67
    Microfluidic formation of coculture tumor spheroids with stromal cells as a novel 3D tumor model for drug testing
    Sun, Qi; Tan, Say Hwa; Chen, Qiushui; Ran, Rui; Hui, Yue; Chen, Dong; Zhao, Chun-Xia
    ACS Biomaterials Science & Engineering (2018), 4 (12), 4425-4433CODEN: ABSEBA; ISSN:2373-9878. (American Chemical Society)
    Three-dimensional (3D) tumor spheroids offer unprecedented capability for drug screening because of their unique features such as spatial 3D structure, relevant physiol. responses, more complex intercellular network, and stroma-cancer cell interactions. Microfluidic technol. provides a facile strategy to make uniform tumor spheroids with potential of high-throughput prodn. In this article, we develop a microfluidic approach to produce core-shell alginate particles, which allows the sep. confinement of different cells in the core and shell structure. To reconstitute the complex tumor structure, we encapsulated tumor cells in the core and stromal fibroblast cells in the shell. These coculture tumor spheroids were applied for drug evaluation showing similar drug resistance as those prepd. using conventional methods in well plates. These results demonstrated that our microfluidic approach are facile and versatile for making various tumor spheroids with uniform size but different components to better mimic tumor microenvironment. Moreover, the prodn. rate of around 200 spheroids/min indicates the great potential of this approach for high-throughput drug screening.
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  68. 68
    Kamperman, T.; Henke, S.; Visser, C. W.; Karperien, M.; Leijten, J. Centering Single Cells in Microgels via Delayed Crosslinking Supports Long-Term 3D Culture by Preventing Cell Escape. Small 2017, 13, 1603711,  DOI: 10.1002/smll.201603711
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    Lee, J. M.; Choi, J. W.; Ahrberg, C. D.; Choi, H. W.; Ha, J. H.; Mun, S. G.; Mo, S. J.; Chung, B. G. Generation of Tumor Spheroids Using a Droplet-Based Microfluidic Device for Photothermal Therapy. Microsystems Nanoeng. 2020,  DOI: 10.1038/s41378-020-0167-x
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    Imaninezhad, M.; Hill, L.; Kolar, G.; Vogt, K.; Zustiak, S. P. Templated Macroporous Polyethylene Glycol Hydrogels for Spheroid and Aggregate Cell Culture. Bioconjugate Chem. 2019, 30, 34,  DOI: 10.1021/acs.bioconjchem.8b00596
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    70
    Templated Macroporous Polyethylene Glycol Hydrogels for Spheroid and Aggregate Cell Culture
    Imaninezhad, Mozhdeh; Hill, Lindsay; Kolar, Grant; Vogt, Kyle; Zustiak, Silviya Petrova
    Bioconjugate Chemistry (2019), 30 (1), 34-46CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
    Macroporous cell-laden hydrogels have recently gained recognition for a wide range of biomedical and bioengineering applications. There are various approaches to create porosity in hydrogels, including lyophilization or foam formation. However, many do not allow a precise control over pore size or are not compatible with in situ cell encapsulation. Here, we developed novel templated macroporous hydrogels by encapsulating uniform degradable hydrogel microspheres produced via microfluidics into a hydrogel slab. The microspheres degraded completely leaving macropores behind. Microsphere degrdn. was dependent on the incubation medium, microsphere size, microsphere confinement in the hydrogel as well as cell encapsulation. Uniquely, the degradable microspheres were biocompatible and when laden with cells, the cells were deposited in the macropores upon microsphere degrdn. and formed multicellular aggregates. The hydrogel-encapsulated cell aggregates were used in a small drug screen to demonstrate the relevance of cell-matrix interactions for multicellular spheroid drug responsiveness. Hydrogel-grown spheroid cultures are increasingly important in applications such as in vitro tumor, hepatocellular, and neurosphere cultures and drug screening; hence, the templated cell aggregate-laden hydrogels described here would find utility in various applications.
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    Hou, Y.; Xie, W.; Achazi, K.; Cuellar-Camacho, J. L.; Melzig, M. F.; Chen, W.; Haag, R. Injectable Degradable PVA Microgels Prepared by Microfluidic Technology for Controlled Osteogenic Differentiation of Mesenchymal Stem Cells. Acta Biomater. 2018, 77, 28,  DOI: 10.1016/j.actbio.2018.07.003
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    71
    Injectable degradable PVA microgels prepared by microfluidic technology for controlled osteogenic differentiation of mesenchymal stem cells
    Hou, Yong; Xie, Wenyan; Achazi, Katharina; Cuellar-Camacho, Jose Luis; Melzig, Matthias F.; Chen, Wei; Haag, Rainer
    Acta Biomaterialia (2018), 77 (), 28-37CODEN: ABCICB; ISSN:1742-7061. (Elsevier Ltd.)
    The direct injection of bone marrow mesenchymal stem cells (hMSCs) is a promising strategy for bone tissue engineering applications. Herein, we have developed injectable degradable poly(vinyl alc.) (PVA) microgels loaded with hMSCs and growth factors and prepd. by a high-throughput microfluidic technol. The PVA-based microgels with tunable mech. and degradable properties were composed of vinyl ether acrylate-functionalized PVA (PVA-VEA) and thiolated PVA-VEA (PVA-VEA-SH) through a Michael-type crosslinking reaction under mild conditions. The hMSCs sustain high viability in PVA microgels, and cell proliferation and migration behaviors can easily be adjusted by varying crosslinking densities of PVA microgels. Addnl., bone morphogenetic protein-2 (BMP-2) co-encapsulated into the microgel environments enhanced osteogenic differentiation of hMSCs as indicated by a significant increase in alk. phosphatase activity, calcium content, and Runx2 and OPN gene expression levels. These results demonstrate the degradable PVA microgels with tailored stem cell microenvironments and controlled release profile of the growth factor to promote and direct differentiation. These PVA-based microgels have promising potential as ideal cell vehicles for applications in regenerative medicine. Stem cell transplantation by an injectable, minimally invasive method has great and promising potential for various injuries, diseases, and tissue regeneration. However, its applications are largely limited owing to the low cell retention and engraftment at the lesion location after administration. We have developed an injectable degradable poly(vinyl alc.) (PVA) microgel prepd. by a high-throughput microfluidic technol. and co-loaded with bone marrow mesenchymal stem cells (hMSCs) and growth factor to protect the stem cells from harsh environmental stress and realize controlled cell differentiation in well-defined microenvironments for bone regeneration. We demonstrated that these degradable PVA microgels can be used as stem cell scaffolds with tailored cell microenvironments and controlled release profile of growth factor to promote and direct differentiation. We are convinced that these PVA-based microgels have promising potential in the future as cellular scaffolds for applications in regenerative medicine.
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  72. 72
    Sabhachandani, P.; Motwani, V.; Cohen, N.; Sarkar, S.; Torchilin, V.; Konry, T. Generation and Functional Assessment of 3D Multicellular Spheroids in Droplet Based Microfluidics Platform. Lab Chip 2016, 16, 497,  DOI: 10.1039/C5LC01139F
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    72
    Generation and functional assessment of 3D multicellular spheroids in droplet based microfluidics platform
    Sabhachandani, P.; Motwani, V.; Cohen, N.; Sarkar, S.; Torchilin, V.; Konry, T.
    Lab on a Chip (2016), 16 (3), 497-505CODEN: LCAHAM; ISSN:1473-0189. (Royal Society of Chemistry)
    Here we describe a robust, microfluidic technique to generate and analyze 3D tumor spheroids, which resembles tumor microenvironment and can be used as a more effective preclin. drug testing and screening model. Monodisperse cell-laden alginate droplets were generated in polydimethylsiloxane (PDMS) microfluidic devices that combine T-junction droplet generation and external gelation for spheroid formation. The proposed approach has the capability to incorporate multiple cell types. For the purposes of our study, we generated spheroids with breast cancer cell lines (MCF-7 drug sensitive and resistant) and co-culture spheroids of MCF-7 together with a fibroblast cell line (HS-5). The device has the capability to house 1000 spheroids on chip for drug screening and other functional anal. Cellular viability of spheroids in the array part of the device was maintained for two weeks by continuous perfusion of complete media into the device. The functional performance of our 3D tumor models and a dose dependent response of std. chemotherapeutic drug, doxorubicin (Dox) and std. drug combination Dox and paclitaxel (PCT) was analyzed on our chip-based platform. Altogether, our work provides a simple and novel, in vitro platform to generate, image and analyze uniform, 3D monodisperse alginate hydrogel tumors for various omic studies and therapeutic efficiency screening, an important translational step before in vivo studies.
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    Langer, K.; Joensson, H. N. Rapid Production and Recovery of Cell Spheroids by Automated Droplet Microfluidics. SLAS Technol. 2020, 25, 111,  DOI: 10.1177/2472630319877376
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    Rapid production and recovery of cell spheroids by automated droplet microfluidics
    Langer, Krzysztof; Joensson, Haakan N.
    SLAS Technology (2020), 25 (2), 111-122CODEN: STLECS; ISSN:2472-6303. (Sage Publications)
    The future of the life sciences is linked to automation and microfluidics. As robots start working side by side with scientists, robotic automation of microfluidics in general, and droplet microfluidics in particular, will significantly extend and accelerate the life sciences. Here, we demonstrate the automation of droplet microfluidics using an inexpensive liq.-handling robot to produce human scaffold-free cell spheroids at high throughput. We use pipet actuation and interface the pipetting tip with a droplet-generating microfluidic device. In this device, we produce highly monodisperse droplets with a diam. coeff. of variation (CV) lower than 2%. By encapsulating cells in these droplets, we produce cell spheroids in droplets and recover them to std. labware containers at a throughput of 85,000 spheroids per microfluidic circuit per h. The viability of the cells in spheroids remains high throughout the process and decreases by >10% (depending on the cell line used) after a 16 h incubation period in nanoliter droplets and automated recovery. Scaffold-free cell spheroids and 3D tissue constructs recapitulate many aspects of functional human tissue more accurately than 2D or single-cell cultures, but assembly methods for spheroids (e.g., hanging drop microplates) have limited throughput. The increased throughput and decreased cost of our method enable spheroid prodn. at the scale needed for lead discovery drug screening, and approach the cost at which these microtissues could be used as building blocks for organ-scale regenerative medicine.
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  74. 74
    Eydelnant, I. A.; Betty Li, B.; Wheeler, A. R. Microgels On-Demand. Nat. Commun. 2014,  DOI: 10.1038/ncomms4355
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  75. 75
    Au, S. H.; Chamberlain, M. D.; Mahesh, S.; Sefton, M. V.; Wheeler, A. R. Hepatic Organoids for Microfluidic Drug Screening. Lab Chip 2014, 14, 3290,  DOI: 10.1039/C4LC00531G
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    75
    Hepatic organoids for microfluidic drug screening
    Au, Sam H.; Chamberlain, M. Dean; Mahesh, Shruthi; Sefton, Michael V.; Wheeler, Aaron R.
    Lab on a Chip (2014), 14 (17), 3290-3299CODEN: LCAHAM; ISSN:1473-0189. (Royal Society of Chemistry)
    We introduce the microfluidic organoids for drug screening (MODS) platform, a digital microfluidic system that is capable of generating arrays of individually addressable, free-floating, three-dimensional hydrogel-based microtissues (or 'organoids'). Here, we focused on liver organoids, driven by the need for early-stage screening methods for hepatotoxicity that enable a "fail early, fail cheaply" strategy in drug discovery. We demonstrate that arrays of hepatic organoids can be formed from co-cultures of HepG2 and NIH-3T3 cells embedded in hydrogel matrixes. The organoids exhibit fibroblast-dependent contractile behavior, and their albumin secretion profiles and cytochrome P 450 3A4 activities are better mimics of in vivo liver tissue than comparable two-dimensional cell culture systems. As proof of principle for screening, MODS was used to generate and analyze the effects of a diln. series of acetaminophen on apoptosis and necrosis. With further development, we propose that the MODS platform may be a cost-effective tool in a "fail early, fail cheaply" paradigm of drug development.
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  76. 76
    Aijian, A. P.; Garrell, R. L. Digital Microfluidics for Automated Hanging Drop Cell Spheroid Culture. J. Lab. Autom. 2015, 20, 283,  DOI: 10.1177/2211068214562002
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    76
    Digital microfluidics for automated hanging drop cell spheroid culture
    Aijian, Andrew P.; Garrell, Robin L.
    Journal of Laboratory Automation (2015), 20 (3), 283-295CODEN: JLAOBH ISSN:. (Sage Publications)
    Cell spheroids are multicellular aggregates, grown in vitro, that mimic the three-dimensional morphol. of physiol. tissues. Although there are numerous benefits to using spheroids in cell-based assays, the adoption of spheroids in routine biomedical research has been limited, in part, by the tedious workflow assocd. with spheroid formation and anal. Here we describe a digital microfluidic platform that has been developed to automate liq.-handling protocols for the formation, maintenance, and anal. of multicellular spheroids in hanging drop culture. We show that droplets of liq. can be added to and extd. from through-holes, or "wells," and fabricated in the bottom plate of a digital microfluidic device, enabling the formation and assaying of hanging drops. Using this digital microfluidic platform, spheroids of mouse mesenchymal stem cells were formed and maintained in situ for 72 h, exhibiting good viability (>90%) and size uniformity (% coeff. of variation <10% intraexperiment, <20% interexperiment). A proof-of-principle drug screen was performed on human colorectal adenocarcinoma spheroids to demonstrate the ability to recapitulate physiol. relevant phenomena such as insulin-induced drug resistance. With automatable and flexible liq. handling, and a wide range of in situ sample prepn. and anal. capabilities, the digital microfluidic platform provides a viable tool for automating cell spheroid culture and anal.
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  77. 77
    Lee, J. M.; Park, D. Y.; Yang, L.; Kim, E. J.; Ahrberg, C. D.; Lee, K. B.; Chung, B. G. Generation of Uniform-Sized Multicellular Tumor Spheroids Using Hydrogel Microwells for Advanced Drug Screening. Sci. Rep. 2018,  DOI: 10.1038/s41598-018-35216-7
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  78. 78
    Chen, Z.; Kheiri, S.; Gevorkian, A.; Young, E. W. K.; Andre, V.; Deisenroth, T.; Kumacheva, E. Microfluidic Arrays of Dermal Spheroids: A Screening Platform for Active Ingredients of Skincare Products. Lab Chip 2021, 21, 3952,  DOI: 10.1039/D1LC00619C
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    78
    Microfluidic arrays of dermal spheroids: a screening platform for active ingredients of skincare products
    Chen, Zhengkun; Kheiri, Sina; Gevorkian, Albert; Young, Edmond W. K.; Andre, Valerie; Deisenroth, Ted; Kumacheva, Eugenia
    Lab on a Chip (2021), 21 (20), 3952-3962CODEN: LCAHAM; ISSN:1473-0189. (Royal Society of Chemistry)
    Organotypic micrometer-size 3D aggregates of skin cells (multicellular spheroids) have emerged as a promising in vitro model that can be utilized as an alternative of animal models to test active ingredients (AIs) of skincare products; however, a reliable dermal spheroid-based microfluidic (MF) model with a goal of in vitro AI screening is yet to be developed. Here, we report a MF platform for the growth of massive arrays of dermal fibroblast spheroids (DFSs) in a biomimetic hydrogel under close-to-physiol. flow conditions and with the capability of screening AIs for skincare products. The DFSs formed after two days of on-chip culture and, in a case study, were used in a time-efficient manner for screening the effect of vitamin C on the synthesis of collagen type I and fibronectin. The computational simulation showed that the uptake of vitamin C was dominated by the advection flux. The results of screening the benchmark AI, vitamin C, proved that DFSs can serve as a reliable in vitro dermal model. The proposed DFS-based MF platform offers a high screening capacity for AIs of skincare products, as well as drug discovery and development in dermatol.
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  79. 79
    Gong, X.; Lin, C.; Cheng, J.; Su, J.; Zhao, H.; Liu, T.; Wen, X.; Zhao, P. Generation of Multicellular Tumor Spheroids with Microwell-Based Agarose Scaffolds for Drug Testing. PLoS One 2015, 10, e0130348,  DOI: 10.1371/journal.pone.0130348
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    79
    Generation of multicellular tumor spheroids with microwell-based agarose scaffolds for drug testing
    Gong, Xue; Lin, Chao; Cheng, Jian; Su, Jiansheng; Zhao, Hang; Liu, Tianlin; Wen, Xuejun; Zhao, Peng
    PLoS One (2015), 10 (6), e0130348/1-e0130348/18CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
    Three dimensional multicellular aggregate, also referred to as cell spheroid or microtissue, is an indispensable tool for in vitro evaluating antitumor activity and drug efficacy. Compared with classical cellular monolayer, multicellular tumor spheroid (MCTS) offers a more rational platform to predict in vivo drug efficacy and toxicity. Nevertheless, traditional processing methods such as plastic dish culture with nonadhesive surfaces are regularly time-consuming, laborious and difficult to provide uniform-sized spheroids, thus causing poor reproducibility of exptl. data and impeding high-throughput drug screening. In order to provide a robust and effective platform for in vitro drug evaluation, we present an agarose scaffold prepd. with the template contg. uniform-sized micro-wells in com. available cell culture plates. The agarose scaffold allows for good adjustment of MCTS size and large-scale prodn. of MCTS. Transparent agarose scaffold also allows for monitoring of spheroid formation under an optical microscopy. The formation of MCTS from MCF-7 cells was prepd. using different-size-well templates and systematically investigated in terms of spheroid growth curve, circularity, and cell viability. The doxorubicin cytotoxicity against MCF-7 spheroid and MCF-7 monolayer cells was compared. The drug penetration behavior, cell cycle distribution, cell apoptosis, and gene expression were also evaluated in MCF-7 spheroid. The findings of this study indicate that, compared with cellular monolayer, MCTS provides a valuable platform for the assessment of therapeutic candidates in an in vivo-mimic microenvironment, and thus has great potential for use in drug discovery and tumor biol. research.
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  80. 80
    Azizipour, N.; Avazpour, R.; Weber, M. H.; Sawan, M.; Ajji, A.; Rosenzweig, D. H. Uniform Tumor Spheroids on Surface-Optimized Microfluidic Biochips for Reproducible Drug Screening and Personalized Medicine. Micromachines 2022, 13 (4), 587,  DOI: 10.3390/mi13040587
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    Mulholland, T.; McAllister, M.; Patek, S.; Flint, D.; Underwood, M.; Sim, A.; Edwards, J.; Zagnoni, M. Drug Screening of Biopsy-Derived Spheroids Using a Self-Generated Microfluidic Concentration Gradient. Sci. Rep. 2018,  DOI: 10.1038/s41598-018-33055-0
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  82. 82
    Huang, C. K.; Paylaga, G. J.; Bupphathong, S.; Lin, K. H. Spherical Microwell Arrays for Studying Single Cells and Microtissues in 3D Confinement. Biofabrication 2020, 12, 025016,  DOI: 10.1088/1758-5090/ab6eda
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    82
    Spherical microwell arrays for studying single cells and microtissues in 3D confinement
    Huang, Cheng-Kuang; Paylaga, Giovanni J.; Bupphathong, Sasinan; Lin, Keng-Hui
    Biofabrication (2020), 12 (2), 025016CODEN: BIOFFN; ISSN:1758-5090. (IOP Publishing Ltd.)
    Microwell arrays have emerged as three-dimensional substrates for cell culture due to their simplicity of fabrication and promise for high-throughput applications such as 3D cell-based assays for drug screening. Motivated by our previous findings that cells display 3D physiol. characteristics when grown in the spherical micropores of monodisperse foam scaffolds, here we engineered novel microwells shaped as spherical caps with obtuse polar angles, yielding narrow apertures. When used as bare substrates, these microwells were suitable for culturing cell spheroids; the narrow apertures sterically hindered unattached cultured cells from rolling out of microwells under agitation. Epithelial cells proliferated and burst out of the aperture, and cell polarity was oriented based on the distribution of extracellular matrix proteins in the microwells. Surprisingly, single fibroblast cells in spherical wells of various diams. (40-100μm) underwent cell-cycle arrest, while cells in circular cylindrical microwells continued to proliferate. Spatial confinement was not sufficient to cause cell-cycle arrest; however, confinement in a const. neg.-curvature microenvironment led to cell-cycle arrest. Overall, these investigations demonstrate that this spherical microwell substrate constitutes a novel basic research tool for elucidating how cells respond to dimensionality and microenvironment with radii of curvature at the cellular length scale.
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  83. 83
    Khot, M. I.; Levenstein, M. A.; de Boer, G. N.; Armstrong, G.; Maisey, T.; Svavarsdottir, H. S.; Andrew, H.; Perry, S. L.; Kapur, N.; Jayne, D. G. Characterising a PDMS Based 3D Cell Culturing Microfluidic Platform for Screening Chemotherapeutic Drug Cytotoxic Activity. Sci. Rep. 2020,  DOI: 10.1038/s41598-020-72952-1
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    Lim, W.; Park, S. A Microfluidic Spheroid Culture Device with a Concentration Gradient Generator for High-Throughput Screening of Drug Efficacy. Molecules 2018, 23, 3355,  DOI: 10.3390/molecules23123355
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    84
    A microfluidic spheroid culture device with a concentration gradient generator for high-throughput screening of drug efficacy
    Lim, Wanyoung; Park, Sungsu
    Molecules (2018), 23 (12), 3355/1-3355/10CODEN: MOLEFW; ISSN:1420-3049. (MDPI AG)
    Three-dimensional (3D) cell culture is considered more clin. relevant in mimicking the structural and physiol. conditions of tumors in vivo compared to two-dimensional cell cultures. In recent years, high-throughput screening (HTS) in 3D cell arrays has been extensively used for drug discovery because of its usability and applicability. Herein, we developed a microfluidic spheroid culture device (muFSCD) with a concn. gradient generator (CGG) that enabled cells to form spheroids and grow in the presence of cancer drug gradients. The device is composed of concave microwells with several serpentine micro-channels which generate a concn. gradient. Once the colon cancer cells (HCT116) formed a single spheroid (approx. 120 mum in diam.) in each microwell, spheroids were perfused in the presence of the cancer drug gradient irinotecan for three days. The no. of spheroids, roundness, and cell viability, were inversely proportional to the drug concn. These results suggest that the muFSCD with a CGG has the potential to become an HTS platform for screening the efficacy of cancer drugs.
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  85. 85
    Pitingolo, G.; Nizard, P.; Riaud, A.; Taly, V. Beyond the on/off Chip Trade-off: A Reversibly Sealed Microfluidic Platform for 3D Tumor Microtissue Analysis. Sensors Actuators, B Chem. 2018, 274, 393,  DOI: 10.1016/j.snb.2018.07.166
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    85
    Beyond the on/off chip trade-off: A reversibly sealed microfluidic platform for 3D tumor microtissue analysis
    Pitingolo, Gabriele; Nizard, Philippe; Riaud, Antoine; Taly, Valerie
    Sensors and Actuators, B: Chemical (2018), 274 (), 393-401CODEN: SABCEB; ISSN:0925-4005. (Elsevier B.V.)
    Nowadays, microfluidic 3D cell culture is widely used to mimic complex microtissue and dynamic environment, performing more realistic in vitro assays for drug testing. Herein, we developed a novel microfluidic platform for tumor microtissue culture, drug response anal. and versatile microscopic characterization. By reversibly bonding the chip, we go beyond the on/off chip tradeoff, which allows us to perform both fluorescence and SEM characterization of tumor microtissues on a simple platform. The microfluidic chip consists of spherical microwells connected via microchannels, bonded through a magnetic system. Colorectal cancer HT-29 cells were cultured as spherical microtissues on chip and their growth kinetics monitored. The cytotoxic activity of Camptothecin was evaluated by in situ live/dead fluorescence staining and quantification of morphol. parameters. Finally, we demonstrated the possibility to collect the 3D tumor microtissues and characterize their surface damaged by the drug using SEM. This reversibly sealed microfluidic platform thus enables to grow sets of 3D tumor microtissues in a controlled dynamic microenvinroment, and subsequently to retrieve the 3D tumor microtissues after chemotherapeutic treatment for in-depth anal.
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  86. 86
    Seyfoori, A.; Samiei, E.; Jalili, N.; Godau, B.; Rahmanian, M.; Farahmand, L.; Majidzadeh-A, K.; Akbari, M. Self-Filling Microwell Arrays (SFMAs) for Tumor Spheroid Formation. Lab Chip 2018, 18, 3516,  DOI: 10.1039/C8LC00708J
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    86
    Self-filling microwell arrays (SFMAs) for tumor spheroid formation
    Seyfoori, Amir; Samiei, Ehsan; Jalili, Neda; Godau, Brent; Rahmanian, Mehdi; Farahmand, Leila; Majidzadeh-A, Keivan; Akbari, Mohsen
    Lab on a Chip (2018), 18 (22), 3516-3528CODEN: LCAHAM; ISSN:1473-0189. (Royal Society of Chemistry)
    Tumor spheroid formation in microwell arrays is a promising approach for high-throughput screening of chemotherapeutic agents. This method offers the advantage of better mimicking the complexities of tumors as compared to conventional monolayer culture systems. However, using these technologies to their full potential is hindered by the inability to seed the cells within the wells uniformly and with high yield and reproducibility. Moreover, std. manufg. approaches for fabrication of microwell arrays rely on lithog. and etching techniques, which are costly, labor-intensive, and time-consuming. Herein, we report on the development of self-filling microwell arrays (SFMAs) in which cells are directed from a loading chamber to microwells using inclined guiding channels. The SFMAs are fabricated by replica molding of three-dimensionally (3D) printed molds in agarose. We characterize the fabrication process, demonstrate the ability to culture breast adenocarcinoma MCF-7 and glioma U87 in SFMAs and perform drug toxicity studies. We envision that the proposed innovative approach opens avenues of opportunities for high-throughput three-dimensional cell culture for drug screening and disease modeling.
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  87. 87
    Bhise, N. S; Manoharan, V.; Massa, S.; Tamayol, A.; Ghaderi, M.; Miscuglio, M.; Lang, Q.; Shrike Zhang, Y.; Shin, S. R.; Calzone, G.; Annabi, N.; Shupe, T. D; Bishop, C. E; Atala, A.; Dokmeci, M. R; Khademhosseini, A. A Liver-on-a-Chip Platform with Bioprinted Hepatic Spheroids. Biofabrication 2016, 8, 014101,  DOI: 10.1088/1758-5090/8/1/014101
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    87
    A liver-on-a-chip platform with bioprinted hepatic spheroids
    Bhise, Nupura S.; Manoharan, Vijayan; Massa, Solange; Tamayol, Ali; Ghaderi, Masoumeh; Miscuglio, Mario; Lang, Qi; Zhang, Yu Shrike; Shin, Su Ryon; Calzone, Giovanni; Annabi, Nasim; Shupe, Thomas D.; Bishop, Colin E.; Atala, Anthony; Dokmeci, Mehmet R.; Khademhosseini, Ali
    Biofabrication (2016), 8 (1), 014101/1-014101/12CODEN: BIOFFN; ISSN:1758-5090. (IOP Publishing Ltd.)
    The inadequacy of animal models in correctly predicting drug and biothreat agent toxicity in humans has resulted in a pressing need for in vitro models that can recreate the in vivo scenario. One of the most important organs in the assessment of drug toxicity is liver. Here, we report the development of a liver-on-a-chip platform for long-term culture of three-dimensional (3D) human HepG2/C3A spheroids for drug toxicity assessment. The bioreactor design allowed for in situ monitoring of the culture environment by enabling direct access to the hepatic construct during the expt. without compromising the platform operation. The engineered bioreactor could be interfaced with a bioprinter to fabricate 3D hepatic constructs of spheroids encapsulated within photocrosslinkable gelatin methacryloyl (GelMA) hydrogel. The engineered hepatic construct remained functional during the 30 days culture period as assessed by monitoring the secretion rates of albumin, alpha-1 antitrypsin, transferrin, and ceruloplasmin, as well as immunostaining for the hepatocyte markers, cytokeratin 18, MRP2 bile canalicular protein and tight junction protein ZO-1. Treatment with 15 mM acetaminophen induced a toxic response in the hepatic construct that was similar to published studies on animal and other in vitro models, thus providing a proof-of-concept demonstration of the utility of this liver-on-a-chip platform for toxicity assessment.
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  88. 88
    Zuchowska, A.; Jastrzebska, E.; Chudy, M.; Dybko, A.; Brzozka, Z. Advanced 3D Spheroid Culture for Evaluation of Photodynamic Therapy in Microfluidic System. Procedia Engineering 2016, 168, 403,  DOI: 10.1016/j.proeng.2016.11.184
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    88
    Advanced 3D Spheroid Culture for Evaluation of Photodynamic Therapy in Microfluidic System
    Zuchowska, A.; Jastrzebska, E.; Chudy, M.; Dybko, A.; Brzozka, Z.
    Procedia Engineering (2016), 168 (), 403-406CODEN: PERNBE; ISSN:1877-7058. (Elsevier Ltd.)
    In this paper we present A549 (carcinoma) and MRC-5 (non-malignant) long-term (10 days) spheroids culture in a microfluidic system. Moreover, the evaluation of photodynamic therapy (PDT) procedure with 5-aminolevulinic acid (ALA) on 3D A549 and MRC-5 spheroids culture was performed. It was found that the spheroids are more resistant for ALA/PDT than monolayer culture. Photocytotoxic effect of the tested ALA concn. on A549 spheroids was noticed 8 days after PDT procedure. Selective effect of PDT procedure on carcinoma cells was obsd.
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  89. 89
    Patra, B.; Chen, Y. H.; Peng, C. C.; Lin, S. C.; Lee, C. H.; Tung, Y. C. A Microfluidic Device for Uniform-Sized Cell Spheroids Formation, Culture, Harvesting and Flow Cytometry Analysis. Biomicrofluidics 2013, 7, 054114,  DOI: 10.1063/1.4824480
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    89
    A microfluidic device for uniform-sized cell spheroids formation, culture, harvesting and flow cytometry analysis
    Patra Bishnubrata; Chen Ying-Hua; Peng Chien-Chung; Tung Yi-Chung; Lin Shiang-Chi; Lee Chau-Hwang
    Biomicrofluidics (2013), 7 (5), 54114 ISSN:1932-1058.
    Culture of cells as three-dimensional (3D) aggregates, named spheroids, possesses great potential to improve in vitro cell models for basic biomedical research. However, such cell spheroid models are often complicated, cumbersome, and expensive compared to conventional Petri-dish cell cultures. In this work, we developed a simple microfluidic device for cell spheroid formation, culture, and harvesting. Using this device, cells could form uniformly sized spheroids due to strong cell-cell interactions and the spatial confinement of microfluidic culture chambers. We demonstrated cell spheroid formation and culture in the designed devices using embryonic stem cells, carcinoma cells, and fibroblasts. We further scaled up the device capable of simultaneously forming and culturing 5000 spheroids in a single chip. Finally, we demonstrated harvesting of the cultured spheroids from the device with a simple setup. The harvested spheroids possess great integrity, and the cells can be exploited for further flow cytometry assays due to the ample cell numbers.
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  90. 90
    Fan, Y.; Nguyen, D. T.; Akay, Y.; Xu, F.; Akay, M. Engineering a Brain Cancer Chip for High-Throughput Drug Screening. Sci. Rep. 2016,  DOI: 10.1038/srep25062
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    Trinh, K. T. L.; Le, N. X. T.; Lee, N. Y. Chitosan-Polydopamine Hydrogel Complex: A Novel Green Adhesion Agent for Reversibly Bonding Thermoplastic Microdevice and Its Application for Cell-Friendly Microfluidic 3D Cell Culture. Lab Chip 2020, 20, 3524,  DOI: 10.1039/D0LC00621A
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    91
    Chitosan-polydopamine hydrogel complex: a novel green adhesion agent for reversibly bonding thermoplastic microdevice and its application for cell-friendly microfluidic 3D cell culture
    Trinh, Kieu The Loan; Le, Nguyen Xuan Thanh; Lee, Nae Yoon
    Lab on a Chip (2020), 20 (19), 3524-3534CODEN: LCAHAM; ISSN:1473-0189. (Royal Society of Chemistry)
    We introduce biocompatible CS-polydopamine hydrogel complex as green adhesion agent for reversible bonding of thermoplastics assisted by UV irradn. PMMA substrates were bonded due to covalent bond network formed between amine groups of either CS or pDA in hydrogel complex and aldehyde groups of the oxidized PMMA surface via Schiff-base reaction during UV irradn. Furthermore, the introduced method allowed for reversible bonding, which is highly appropriate for the fabrication of microdevices for cell-related applications. Surface characterizations such as water contact angle measurement, SEM anal., AFM and FTIR were performed to confirm successful coating of hydrogel complex on the PMMA surface. The bonding between two PMMAs or PMMA with other thermoplastics was successfully investigated with high bond strengths ranging from 0.4 to 0.7 MPa. The potential for reversible bonding of this method was verified by repeating the bonding/debonding cycle of the bonded PMMAs for three times, which maintained the bond strength at approx. 0.5 MPa. The compatibility of bonding method in biol. applications was examd. by culturing MSC inside microchannel where multiple uniform-sized MSC spheroids were successfully formed. Then, spheroids were harvested for off-chip expts. enabled by the reversibility of the introduced bonding strategy.
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  92. 92
    Huang, Y. L.; Ma, Y.; Wu, C.; Shiau, C.; Segall, J. E.; Wu, M. Tumor Spheroids under Perfusion within a 3D Microfluidic Platform Reveal Critical Roles of Cell-Cell Adhesion in Tumor Invasion. Sci. Rep. 2020,  DOI: 10.1038/s41598-020-66528-2
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    Zhao, L.; Liu, Y.; Liu, Y.; Zhang, M.; Zhang, X. Microfluidic Control of Tumor and Stromal Cell Spheroids Pairing and Merging for Three-Dimensional Metastasis Study. Anal. Chem. 2020, 92, 7638,  DOI: 10.1021/acs.analchem.0c00408
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    93
    Microfluidic Control of Tumor and Stromal Cell Spheroids Pairing and Merging for Three-Dimensional Metastasis Study
    Zhao, Liang; Liu, Yingying; Liu, Yang; Zhang, Meiqin; Zhang, Xueji
    Analytical Chemistry (Washington, DC, United States) (2020), 92 (11), 7638-7645CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    Three-dimensional cell culture provides an efficient way to simulate the in vivo tumorigenic microenvironment where tumor-stroma interaction intrinsically plays a pivotal role. Conventional three-dimensional (3D) culture is inadequate to address precise coexistential heterogeneous pairing and quant. measurement in a parallel algorithm format. Herein, we implemented a set of microwell array microfluidic devices to study the cell spheroids-based tumor-stromal metastatic process in vitro. This approach enables accurate one-to-one pairing between tumor and fibroblast spheroid for dissecting 3D tumor invasion in the manner of high-content imaging. On one single device, 240 addressable tumor-stroma pairings can be formed with convenient pipetting and centrifugation within a small area of 1 cm2. Consequential confocal imaging anal. disclosed that the tumor spheroid could envelop the fibroblast spheroid. Specific chems. can effectively hamper or promote this 3D metastasis. Due to the addressable time-resolved measurements of the merging process of hundreds of doublets, our approach allows us to decipher the metastatic phenotype between different tumor spheroids. Compared with traditional protocols, massive heterogeneous cellular spheroids pairing and merging using this method is well-defined with microfluidic control, which leads to a favorable high-content tumor-stroma doublet metastasis anal. This simple technique will be a useful tool for investigating heterotypic spheroid-spheroid interactions.
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  94. 94
    Eilenberger, C.; Rothbauer, M.; Selinger, F.; Gerhartl, A.; Jordan, C.; Harasek, M.; Schädl, B.; Grillari, J.; Weghuber, J.; Neuhaus, W.; Küpcü, S.; Ertl, P. A Microfluidic Multisize Spheroid Array for Multiparametric Screening of Anticancer Drugs and Blood-Brain Barrier Transport Properties. Adv. Sci. 2021, 8, 2004856,  DOI: 10.1002/advs.202004856
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    94
    A Microfluidic Multisize Spheroid Array for Multiparametric Screening of Anticancer Drugs and Blood-Brain Barrier Transport Properties
    Eilenberger, Christoph; Rothbauer, Mario; Selinger, Florian; Gerhartl, Anna; Jordan, Christian; Harasek, Michael; Schadl, Barbara; Grillari, Johannes; Weghuber, Julian; Neuhaus, Winfried; Kuepcue, Seta; Ertl, Peter
    Advanced Science (Weinheim, Germany) (2021), 8 (11), 2004856CODEN: ASDCCF; ISSN:2198-3844. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Physiol.-relevant in vitro tissue models with their promise of better predictability have the potential to improve drug screening outcomes in preclin. studies. Despite the advances of spheroid models in pharmaceutical screening applications, variations in spheroid size and consequential altered cell responses often lead to nonreproducible and unpredictable results. Here, a microfluidic multisize spheroid array is established and characterized using liver, lung, colon, and skin cells as well as a triple-culture model of the blood-brain barrier (BBB) to assess the effects of spheroid size on (a) anticancer drug toxicity and (b) compd. penetration across an advanced BBB model. The reproducible on-chip generation of 360 spheroids of five dimensions on a well-plate format using an integrated microlens technol. is demonstrated. While spheroid size-related IC50 values vary up to 160% using the anticancer drugs cisplatin (CIS) or doxorubicin (DOX), reduced CIS:DOX drug dose combinations eliminate all lung microtumors independent of their sizes. A further application includes optimizing cell seeding ratios and size-dependent compd. uptake studies in a perfused BBB model. Generally, smaller BBB-spheroids reveal an 80% higher compd. penetration than larger spheroids while verifying the BBB opening effect of mannitol and a spheroid size-related modulation on paracellular transport properties.
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  95. 95
    Järvinen, P.; Bonabi, A.; Jokinen, V.; Sikanen, T. Simultaneous Culturing of Cell Monolayers and Spheroids on a Single Microfluidic Device for Bridging the Gap between 2D and 3D Cell Assays in Drug Research. Adv. Funct. Mater. 2020, 30, 2000479,  DOI: 10.1002/adfm.202000479
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    Zuchowska, A.; Jastrzebska, E.; Zukowski, K.; Chudy, M.; Dybko, A.; Brzozka, Z. A549 and MRC-5 Cell Aggregation in a Microfluidic Lab-on-a-Chip System. Biomicrofluidics 2017, 11, 024110,  DOI: 10.1063/1.4979104
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    96
    A549 and MRC-5 cell aggregation in a microfluidic Lab-on-a-chip system
    Zuchowska, A.; Jastrzebska, E.; Zukowski, K.; Chudy, M.; Dybko, A.; Brzozka, Z.
    Biomicrofluidics (2017), 11 (2), 024110/1-024110/13CODEN: BIOMGB; ISSN:1932-1058. (American Institute of Physics)
    In this paper, we present a culture of A549 and MRC-5 spheroids in a microfluidic system. The aim of our work was to develop a good lung cancer model for the evaluation of drug cytotoxicity. Our research was focused on detg. the progress of cell aggregation depending on such factors as the depth of culture microwells in the microdevices, a different flow rate of the introduced cell suspensions, and the addn. of collagen to cell suspensions. We showed that these factors had a significant influence on spheroid formation. It was found that both MRC-5 and A549 cells exhibited higher aggregation in 500μm microwells. We also noticed that collagen needs to be added to A549 cells to form the spheroids. Optimizing the mentioned parameters allowed us to form 3D lung tissue models in the microfluidic system during the 10-day culture. This study indicates how important an appropriate selection of the specified parameters is (e.g., geometry of the microwells in the microsystem) to obtain the spheroids characterized by high viability in the microfluidic system. (c) 2017 American Institute of Physics.
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  97. 97
    Hsu, C.-H.; Chen, C.-C. Microfluidic Hanging Drop Chip. TW 201329230 A, 2013.
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    Rodoplu, D.; Matahum, J. S.; Hsu, C.-H. A Microfluidic Hanging Drop-Based Spheroid Co-Culture Platform for Probing Tumor Angiogenesis. Lab Chip 2022, 22, 1275,  DOI: 10.1039/D1LC01177D
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    98
    A microfluidic hanging drop-based spheroid co-culture platform for probing tumor angiogenesis
    Rodoplu, Didem; Matahum, Jefunnie Sierra; Hsu, Chia-Hsien
    Lab on a Chip (2022), 22 (7), 1275-1285CODEN: LCAHAM; ISSN:1473-0189. (Royal Society of Chemistry)
    Co-culturing of embryoid bodies (EBs) and tumor spheroids (TSs) allows mimicking tumor angiogenesis in vitro. Here, we report a microfluidic hanging drop-based spheroid co-culture device (μ-CCD) that permits the generation and co-culturing of EBs and TSs using a simple manual operation procedure and setup. In brief, uniform-sized EBs and TSs can be generated on the device in eight pairs of hanging droplets from adjacent microfluidic channels, followed by the confrontation of EB and TS pairs by merging the droplet pairs to culture the EB-TS spheroids to investigate tumor-induced angiogenic sprouting. The phys. parameters of the device were optimized to maintain the long-term stability of hanging droplets for up to ten days. The mouse embryonic stem cell line ES-D3 and breast cancer cell lines MDA-MB-231 and MCF-7 were used to generate EBs, invasive TSs, and non-invasive TSs resp. Confocal imaging results showed that the vessel percentage area and total vessel length which are linked to tumor angiogenesis increased after 6 days of co-culturing. An anti-angiogenesis drug testing on the co-cultured EB-TS spheroids was also demonstrated in the device. The μ-CCD provides a simple yet high-efficiency method to generate and co-culture cell spheroids and may also be useful for other applications involving spheroid co-culturing.
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  99. 99
    Tung, Y. C.; Hsiao, A. Y.; Allen, S. G.; Torisawa, Y. S.; Ho, M.; Takayama, S. High-Throughput 3D Spheroid Culture and Drug Testing Using a 384 Hanging Drop Array. Analyst 2011, 136, 473,  DOI: 10.1039/C0AN00609B
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    99
    High-throughput 3D spheroid culture and drug testing using a 384 hanging drop array
    Tung, Yi-Chung; Hsiao, Amy Y.; Allen, Steven G.; Torisawa, Yu-suke; Ho, Mitchell; Takayama, Shuichi
    Analyst (Cambridge, United Kingdom) (2011), 136 (3), 473-478CODEN: ANALAO; ISSN:0003-2654. (Royal Society of Chemistry)
    Culture of cells as three-dimensional (3D) aggregates can enhance in vitro tests for basic biol. research as well as for therapeutics development. Such 3D culture models, however, are often more complicated, cumbersome, and expensive than two-dimensional (2D) cultures. This paper describes a 384-well format hanging drop culture plate that makes spheroid formation, culture, and subsequent drug testing on the obtained 3D cellular constructs as straightforward to perform and adapt to existing high-throughput screening (HTS) instruments as conventional 2D cultures. Using this platform, we show that drugs with different modes of action produce distinct responses in the physiol. 3D cell spheroids compared to conventional 2D cell monolayers. Specifically, the anticancer drug 5-fluorouracil (5-FU) has higher anti-proliferative effects on 2D cultures whereas the hypoxia activated drug commonly referred to as tirapazamine (TPZ) are more effective against 3D cultures. The multiplexed 3D hanging drop culture and testing plate provides an efficient way to obtain biol. insights that are often lost in 2D platforms.
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  100. 100
    Wu, H. W.; Hsiao, Y. H.; Chen, C. C.; Yet, S. F.; Hsu, C. H. A Pdms-Based Microfluidic Hanging Drop Chip for Embryoid Body Formation. Molecules 2016, 21, 882,  DOI: 10.3390/molecules21070882
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    100
    A PDMS-based microfluidic hanging drop chip for embryoid body formation
    Wu, Huei-Wen; Hsiao, Yi-Hsing; Chen, Chih-Chen; Yet, Shaw-Fang; Hsu, Chia-Hsien
    Molecules (2016), 21 (7), 882/1-882/11CODEN: MOLEFW; ISSN:1420-3049. (MDPI AG)
    The conventional hanging drop technique is the most widely used method for embryoid body (EB) formation. However, this method is labor intensive and limited by the difficulty in exchanging the medium. Here, we report a microfluidic chip-based approach for high-throughput formation of EBs. The device consists of microfluidic channels with 6 × 12 opening wells in PDMS supported by a glass substrate. The PDMS channels were fabricated by replicating polydimethyl-siloxane (PDMS) from SU-8 mold. The droplet formation in the chip was tested with different hydrostatic pressures to obtain optimal operation pressures for the wells with 1000μm diam. openings. The droplets formed at the opening wells were used to culture mouse embryonic stem cells which could subsequently developed into EBs in the hanging droplets. This device also allows for medium exchange of the hanging droplets making it possible to perform immunochem. staining and characterize EBs on chip.
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  101. 101
    Huang, S. W.; Tzeng, S. C.; Chen, J. K.; Sun, J. S.; Lin, F. H. A Dynamic Hanging-Drop System for Mesenchymal Stem Cell Culture. Int. J. Mol. Sci. 2020, 21, 4298,  DOI: 10.3390/ijms21124298
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    101
    A dynamic hanging-drop system for mesenchymal stem cell culture
    Huang, Shu-Wei; Tzeng, Shian-Chiuan; Chen, Jem-Kun; Sun, Jui-Sheng; Lin, Feng-Huei
    International Journal of Molecular Sciences (2020), 21 (12), 4298CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)
    There have been many microfluid technologies combined with hanging-drop for cell culture gotten developed in the past decade. A common problem within these devices is that the cell suspension introduced at the central inlet could cause a no. of cells in each microwell to not regularize. Also, the instability of droplets during the spheroid formation remains an unsolved ordeal. In this study, we designed a microfluidic-based hanging-drop culture system with the design of taper-tube that can increase the stability of droplets while enhancing the rate of liq. exchange. A ring is surrounding the taper-tube. The ring can hold the cells to enable us to seed an adequate amt. of cells before perfusion. Moreover, during the period of cell culture, the mech. force around the cell is relatively low to prevent stem cells from differentiate and maintain the phenotype. As a result of our hanging system design, cells are designed to accumulate at the bottom of the droplet. This method enhances convenience for observation activities and anal. of expts. Thus, this microfluid chip can be used as an in vitro platform representing in vivo physiol. conditions, and can be useful in regenerative therapy.
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  102. 102
    Cho, C. Y.; Chiang, T. H.; Hsieh, L. H.; Yang, W. Y.; Hsu, H. H.; Yeh, C. K.; Huang, C. C.; Huang, J. H. Development of a Novel Hanging Drop Platform for Engineering Controllable 3D Microenvironments. Front. Cell Dev. Biol. 2020,  DOI: 10.3389/fcell.2020.00327
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    Ganguli, A.; Mostafa, A.; Saavedra, C.; Kim, Y.; Le, P.; Faramarzi, V.; Feathers, R. W.; Berger, J.; Ramos-Cruz, K. P.; Adeniba, O.; Diaz, G. J. P.; Drnevich, J.; Wright, C. L.; Hernandez, A. G.; Lin, W.; Smith, A. M.; Kosari, F.; Vasmatzis, G.; Anastasiadis, P. Z.; Bashir, R. Three-Dimensional Microscale Hanging Drop Arrays with Geometric Control for Drug Screening and Live Tissue Imaging. Sci. Adv. 2021,  DOI: 10.1126/sciadv.abc1323
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    Liu, X.; Lin, H.; Song, J.; Zhang, T.; Wang, X.; Huang, X.; Zheng, C. A Novel Simpledrop Chip for 3d Spheroid Formation and Anti-Cancer Drug Assay. Micromachines 2021, 12, 681,  DOI: 10.3390/mi12060681
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    Sun, B.; Zhao, Y.; Wu, W.; Zhao, Q.; Li, G. A Superhydrophobic Chip Integrated with an Array of Medium Reservoirs for Long-Term Hanging Drop Spheroid Culture. Acta Biomater. 2021, 135, 234,  DOI: 10.1016/j.actbio.2021.08.006
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    105
    A superhydrophobic chip integrated with an array of medium reservoirs for long-term hanging drop spheroid culture
    Sun, Bangyong; Zhao, Yi; Wu, Weimin; Zhao, Qiang; Li, Gang
    Acta Biomaterialia (2021), 135 (), 234-242CODEN: ABCICB; ISSN:1742-7061. (Elsevier Ltd.)
    Hanging drop (HD) is one of the most popular methods used for forming three-dimensional (3D) cell spheroids. However, conventional hanging drop systems are only applicable for short-term spheroid culture due to their inconvenience in exchanging cell culture media. Here we present a medium-reservoir-integrated superhydrophobic (MRI-SH) chip for long-term HD spheroid cultures. The device consists of two main components: i a patterned superhydrophobic (SH) surface contg. an array of wettable spots which anchor arrays of droplets of cell suspension, and ii an array of chambers that serve as medium reservoirs, both interconnected via an array of through-holes. This configuration provides two distinct advantages over conventional HD configurations: i the high wettability contrast of the SH pattern on the chip leads to the formation and adhesion of nearly spherical hanging droplets on its surface, which minimizes interactions between the liq. and the substrate; ii the integrated chambers provide large vols. of medium to maintain longer culture durations. Using this device, spheroids of MHCC97H cells were successfully formed, and the cultured spheroids could maintain high viability for up to 30 days and exhibited enhanced spheroid morphol. compared to those cultured in the conventional HD systems. This paper presents a medium-reservoir-integrated superhydrophobic hanging drop (HD) platform for the long-term culture of spheroids with enhanced morphol. By monolithically integrating medium reservoirs and a patterned SH surface into a single device, this HD platform can not only produce high-quality spheroids, but also permit them to sustain high viability for up to 30 days without the need for tedious medium replenishment. We believe that such a platform will be valuable in a wide range of biol. or biomedical applications, including tissue engineering, regenerative medicine, and drug discovery.
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  106. 106
    Fu, C. Y.; Tseng, S. Y.; Yang, S. M.; Hsu, L.; Liu, C. H.; Chang, H. Y. A Microfluidic Chip with a U-Shaped Microstructure Array for Multicellular Spheroid Formation, Culturing and Analysis. Biofabrication 2014, 6, 015009,  DOI: 10.1088/1758-5082/6/1/015009
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    106
    A microfluidic chip with a U-shaped microstructure array for multicellular spheroid formation, culturing and analysis
    Fu, Chien-Yu; Tseng, Sheng-Yang; Yang, Shih-Mo; Hsu, Long; Liu, Cheng-Hsien; Chang, Hwan-You
    Biofabrication (2014), 6 (1), 015009CODEN: BIOFFN; ISSN:1758-5090. (IOP Publishing Ltd.)
    Multicellular spheroids (MCS), formed by self-assembly of single cells, are commonly used as a three-dimensional cell culture model to bridge the gap between in vitro monolayer culture and in vivo tissues. However, current methods for MCS generation and anal. still suffer drawbacks such as being labor-intensive and of poor controllability, and are not suitable for high-throughput applications. This study demonstrates a novel microfluidic chip to facilitate MCS formation, culturing and anal. The chip contains an array of U-shaped microstructures fabricated by photopolymg. the poly(ethylene glycol) diacrylate hydrogel through defining the UV light exposure pattern with a photomask. The geometry of the U-shaped microstructures allowed trapping cells into the pocket through the actions of fluid flow and the force of gravity. The hydrogel is non-adherent for cells, promoting the formation of MCS. Its permselective property also facilitates exchange of nutrients and waste for MCS, while providing protection of MCS from shearing stress during the medium perfusion. Heterotypic MCS can be formed easily by manipulating the cell trapping steps. Subsequent drug susceptibility anal. and long-term culture could also be achieved within the same chip. This MCS formation and culture platform can be used as a micro-scale bioreactor and applied in many cell biol. and drug testing studies.
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  107. 107
    Torisawa, Y.-s.; Takagi, A.; Nashimoto, Y.; Yasukawa, T.; Shiku, H.; Matsue, T. A Multicellular Spheroid Array to Realize Spheroid Formation, Culture, and Viability Assay on a Chip. Biomaterials 2007, 28, 559,  DOI: 10.1016/j.biomaterials.2006.08.054
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    107
    A multicellular spheroid array to realize spheroid formation, culture, and viability assay on a chip
    Torisawa, Yu-suke; Takagi, Airi; Nashimoto, Yuji; Yasukawa, Tomoyuki; Shiku, Hitoshi; Matsue, Tomokazu
    Biomaterials (2007), 28 (3), 559-566CODEN: BIMADU; ISSN:0142-9612. (Elsevier Ltd.)
    We describe a novel multicellular spheroid culture system that facilitates the easy prepn. and culture of a spheroid microarray for the long-term monitoring of cellular activity. A spheroid culture device with an array of pyramid-like microholes was constructed in a silicon chip that was equipped with elastomeric microchannels. A cell suspension was introduced via the microfluidic channel into the microstructure that comprised silicon microholes and elastomeric microwells. A single spheroid can be formed and localized precisely within each microstructure. Since the culture medium could be replaced via the microchannels, a long-term culture (of ∼2 wk) is available on the chip. Measurement of albumin prodn. in the hepatoma cell line (HepG2) showed that the liver-specific functions were maintained for 2 wk. Based on the cellular respiratory activity, the cellular viability of the spheroid array on the chip was evaluated using scanning electrochem. microscopy. Responses to four different chem. stimulations were simultaneously detected on the same chip, thus demonstrating that each channel could be evaluated independently under various stimulation conditions. Our spheroid culture system facilitated the understanding of spheroid formation, culture, and viability assay on a single chip, thus functioning as a useful drug-screening device for cancer and liver cells.
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  108. 108
    He, Y.; Huang, B.; Rofaani, E.; Hu, J.; Liu, Y.; Pitingolo, G.; Wang, L.; Shi, J.; Aimé, C.; Chen, Y. Fabrication of Micro-Cages and Caged Tumor Spheroids for Microfluidic Chip-Based Assays. Microelectron. Eng. 2020, 225, 111256,  DOI: 10.1016/j.mee.2020.111256
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    108
    Fabrication of micro-cages and caged tumor spheroids for microfluidic chip-based assays
    He, Yong; Huang, Boxin; Rofaani, Elrade; Hu, Jie; Liu, Yuanhui; Pitingolo, Gabriele; Wang, Li; Shi, Jian; Aime, Carole; Chen, Yong
    Microelectronic Engineering (2020), 225 (), 111256CODEN: MIENEF; ISSN:0167-9317. (Elsevier B.V.)
    We developed a simple method to fabricate micro-cages and caged tumor spheroids for microfluidic chip-based assays. The micro-cage device consists of an array of honeycomb compartments with a monolayer of crosslinked and agarose-coated gelatin nanofibers at the bottom and a mesh of 200μm hole-size on the top. U87-MG single cells were dispersed through the mesh and resulted tumor spheroids confined in each of the cage compartment after incubation. As expected, the tumor spheroids are one-by-one distributed in each of the compartment with the same size and they grew inside the compartments. The final size of the spheroid was limited by both diffusion and confinement. If the height of the cage is small, the nanofiber layer underneath tumors could be deflected due to mechanic stress of growing tumors. If the height of the cage is large, tumors grew freely without stress but their size was limited by diffusion. In both cases, tumors tended to remain in spherical shape. To illustrate the robustness of the approach, the tumor caged device was reversibly integrated into a microfluidic chip for drug test. Our results show that under tangent flow conditions, combretastatin A-4 had a clear effect on tumor disassembling.
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  109. 109
    Yu, L.; Chen, M. C. W.; Cheung, K. C. Droplet-Based Microfluidic System for Multicellular Tumor Spheroid Formation and Anticancer Drug Testing. Lab Chip 2010, 10, 2424,  DOI: 10.1039/c004590j
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    109
    Droplet-based microfluidic system for multicellular tumor spheroid formation and anticancer drug testing
    Yu, Linfen; Chen, Michael C. W.; Cheung, Karen C.
    Lab on a Chip (2010), 10 (18), 2424-2432CODEN: LCAHAM; ISSN:1473-0197. (Royal Society of Chemistry)
    Creating multicellular tumor spheroids is crit. for characterizing anticancer treatments since it may provide a better model than monolayer culture of tumor cells. Moreover, continuous dynamic perfusion allows the establishment of long term cell culture and subsequent multicellular spheroid formation. A droplet-based microfluidic system was used to form alginate beads with entrapped breast tumor cells. After gelation, the alginate beads were trapped in microsieve structures for cell culture in a continuous perfusion system. The alginate environment permitted cell proliferation and the formation of multicellular spheroids was obsd. The dose-dependent response of the tumor spheroids to doxorubicin, and anticancer drug, showed multicellular resistance compared to conventional monolayer culture. The microsieve structures maintain const. location of each bead in the same position throughout the device seeding process, cell proliferation and spheroid formation, treatment with drug, and imaging, permitting temporal and spatial tracking.
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    Chen, Y.; Gao, D.; Wang, Y.; Lin, S.; Jiang, Y. A Novel 3D Breast-Cancer-on-Chip Platform for Therapeutic Evaluation of Drug Delivery Systems. Anal. Chim. Acta 2018, 1036, 97,  DOI: 10.1016/j.aca.2018.06.038
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    110
    A novel 3D breast-cancer-on-chip platform for therapeutic evaluation of drug delivery systems
    Chen, Yongli; Gao, Dan; Wang, Yanwei; Lin, Shuo; Jiang, Yuyang
    Analytica Chimica Acta (2018), 1036 (), 97-106CODEN: ACACAM; ISSN:0003-2670. (Elsevier B.V.)
    The ability to rapidly screen drugs and drug delivery systems with a more accurate tumor model to better predict their in vivo performance is of great importance in drug development, because there have been some limitations in currently used tumor models. To address this problem, we developed an in vitro breast tumor model on a chip, composed of a microvessel wall, the extracellular matrix (ECM) and uniformly sized multicellular tumor spheroids (MCTS), for the evaluation of nanoparticle-based drug delivery systems. A carbon dots (CDs)-based drug delivery system was synthesized as a model to evaluate the real-time monitoring ability of the system transport through the endothelium and the penetrability into MCTS with a high spatio-temporal resoln. on the established platform. Moreover, a modified 96-well plate was used to hold the microfluidic devices for in situ cytotoxicity assays of the MCTS by a microplate reader. Our findings revealed that the synthesized drug delivery system could be transported across an endothelial monolayer within 3 h and was nontoxic to the cells throughout the expt. In addn., we demonstrated the capabilities of this model by assessing the delivery and efficacy of the drug delivery system in BT549 and T47D spheroids, two cell lines representative of triple neg. breast cancer (TNBC) and non-TNBC, resp. This microfluidic platform enables evaluation of dynamic transport behavior and in situ cytotoxicity evaluation in one system. The established platform provides a more accurate and low-cost in vitro model for rapid drug screening in pre-clin. studies.
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  111. 111
    Hardelauf, H.; Frimat, J. P.; Stewart, J. D.; Schormann, W.; Chiang, Y. Y.; Lampen, P.; Franzke, J.; Hengstler, J. G.; Cadenas, C.; Kunz-Schughart, L. A.; West, J. Microarrays for the Scalable Production of Metabolically Relevant Tumour Spheroids: A Tool for Modulating Chemosensitivity Traits. Lab Chip 2011, 11, 419,  DOI: 10.1039/C0LC00089B
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    111
    Microarrays for the scalable production of metabolically relevant tumor spheroids: a tool for modulating chemosensitivity traits
    Hardelauf, Heike; Frimat, Jean-Philippe; Stewart, Joanna D.; Schormann, Wiebke; Chiang, Ya-Yu; Lampen, Peter; Franzke, Joachim; Hengstler, Jan G.; Cadenas, Cristina; Kunz-Schughart, Leoni A.; West, Jonathan
    Lab on a Chip (2011), 11 (3), 419-428CODEN: LCAHAM; ISSN:1473-0197. (Royal Society of Chemistry)
    We report the use of thin film poly(dimethylsiloxane) (PDMS) prints for the arrayed mass prodn. of highly uniform 3-D human HT29 colon carcinoma spheroids. The spheroids have an organotypic d. and, as detd. by 3-axis imaging, were genuinely spherical. Critically, the array d. impacts growth kinetics and can be tuned to produce spheroids ranging in diam. from 200 to 550 μm. The diffusive limit of competition for media occurred with a pitch of ≥1250 μm and was used for the optimal array-based culture of large, viable spheroids. During sustained culture mass transfer gradients surrounding and within the spheroids are established, and lead to growth cessation, altered expression patterns and the formation of a central secondary necrosis. These features reflect the microenvironment of avascularised tumors, making the array format well suited for the prodn. of model tumors with defined sizes and thus defined spatio-temporal pathophysiol. gradients. Exptl. windows, before and after the onset of hypoxia, were identified and used with an enzyme activity-based viability assay to measure the chemosensitivity towards irinotecan. Compared to monolayer cultures, a marked redn. in the drug efficacy towards the different spheroid culture states was obsd. and attributed to cell cycle arrest, the 3-D character, scale and/or hypoxia factors. In summary, spheroid culture using the array format has great potential to support drug discovery and development, as well as tumor biol. research.
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  112. 112
    Barisam, M.; Niavol, F. R.; Kinj, M. A.; Saidi, M. S.; Ghanbarian, H.; Kashaninejad, N. Enrichment of Cancer Stem-like Cells by Controlling Oxygen, Glucose and Fluid Shear Stress in a Microfluidic Spheroid Culture Device. J. Sci. Adv. Mater. Devices 2022, 7, 100439,  DOI: 10.1016/j.jsamd.2022.100439
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    112
    Enrichment of cancer stem-like cells by controlling oxygen, glucose and fluid shear stress in a microfluidic spheroid culture device
    Barisam, Maryam; Niavol, Fazeleh Ranjbar; Kinj, Moslem Afrasiabi; Saidi, Mohammad Said; Ghanbarian, Hossein; Kashaninejad, Navid
    Journal of Science: Advanced Materials and Devices (2022), 7 (2), 100439CODEN: JSAMCR; ISSN:2468-2179. (Elsevier B.V.)
    Current chemotherapies can often kill fast-growing cancer cells but are ineffective in destroying cancer stem cells (CSCs). This study aimed to investigate the effect of in vitro cell culture conditions on the features of lung adenocarcinoma cells (represented by A549, non-small cell type (NSCLC), cell lines) and the induction of cancer stem-like cells. For this purpose, a microfluidic system contg. U-shaped arrays was designed and numerically optimized. This system was used for the three-dimensional culture of cells under a continuous laminar flow of culture medium. Numerical simulations were also performed to est. the quiescent and necrotic zones inside the spheroids and the fluid shear stress on the cultured spheroids. Moreover, the effects of culture medium flow rate, oxygen and glucose concns. and anoikis phenomenon on the no. of CD90+ cells and expression of stemness, epithelial-mesenchymal transition (EMT) and ATP-binding cassette (ABC)-transporters genes were investigated. The results showed that all these parameters substantially affected the enrichment of A549 cancer stem-like cells. We also investigated the effect of the CSC enrichment method, i.e., shear stress and oxygen and glucose concns., on resistance to cisplatin treatment. Increasing shear stress and glucose concn. and decreasing oxygen concn. led to a sharp increase in chemoresistance of cells. Interestingly, changing oxygen concn. was more significant than changing the other factors. The results of this paper can be helpful for the effective enrichment of CSCs by adjusting in vitro culture conditions.
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  113. 113
    Dornhof, J.; Kieninger, J.; Muralidharan, H.; Maurer, J.; Urban, G. A.; Weltin, A. Oxygen and Lactate Monitoring in 3D Breast Cancer Organoid Culture with Sensor-Integrated Microfluidic Platform. 21st Int. Conf. Solid-State Sensors, Actuators Microsystems, TRANSDUCERS 2021 2021, June, 703– 706 DOI: 10.1109/Transducers50396.2021.9495557
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    Chen, B.; Wu, Y.; Ao, Z.; Cai, H.; Nunez, A.; Liu, Y.; Foley, J.; Nephew, K.; Lu, X.; Guo, F. High-Throughput Acoustofluidic Fabrication of Tumor Spheroids. Lab Chip 2019, 19, 1755,  DOI: 10.1039/C9LC00135B
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    114
    High-throughput acoustofluidic fabrication of tumor spheroids
    Chen, Bin; Wu, Yue; Ao, Zheng; Cai, Hongwei; Nunez, Asael; Liu, Yunhua; Foley, John; Nephew, Kenneth; Lu, Xiongbin; Guo, Feng
    Lab on a Chip (2019), 19 (10), 1755-1763CODEN: LCAHAM; ISSN:1473-0189. (Royal Society of Chemistry)
    Three-dimensional (3D) culture of multicellular spheroids, offering a desirable biomimetic microenvironment, is appropriate for recapitulating tissue cellular adhesive complexity and revealing a more realistic drug response. However, current 3D culture methods are suffering from low-throughput, poor controllability, intensive-labor, and variation in spheroid size, thus not ready for many high-throughput screening applications including drug discovery and toxicity testing. Herein, we developed a high-throughput multicellular spheroid fabrication method using acoustofluidics. By acoustically-assembling cancer cells with low-cost and disposable devices, our method can produce more than 12 000 multicellular aggregates within several minutes and allow us to transfer these aggregates into ultra-low attachment dishes for long-term culture. This method can generate more than 6000 tumor spheroids per operation, and reduce tumor spheroid formation time to one day. Our platform has advantages in forming spheroids with high throughput, short time, and long-term effectiveness, and is easy-to-operation. This acoustofluidic spheroid assembly method provides a simple and efficient way to produce large nos. of uniform-sized spheroids for biomedical applications in translational medicine, pharmaceutical industry and basic life science research.
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  115. 115
    Wu, Z.; Chen, B.; Wu, Y.; Xia, Y.; Chen, H.; Gong, Z.; Hu, H.; Ding, Z.; Guo, S. Scaffold-Free Generation of Heterotypic Cell Spheroids Using Acoustofluidics. Lab Chip 2021, 21 (18), 3498– 3508,  DOI: 10.1039/D1LC00496D
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    115
    Scaffold-free generation of heterotypic cell spheroids using acoustofluidics
    Wu, Zhuhao; Chen, Bin; Wu, Yue; Xia, Yu; Chen, Hui; Gong, Zhiyi; Hu, Hang; Ding, Zhao; Guo, Shishang
    Lab on a Chip (2021), 21 (18), 3498-3508CODEN: LCAHAM; ISSN:1473-0189. (Royal Society of Chemistry)
    3D Cell cultures such as cell spheroids are widely used for tissue engineering, regenerative medicine, and translational medicine, but challenges remain in recapitulating the architectural complexity and spatiotemporal heterogeneity of tissues. Thus, we developed a scaffold-free and versatile acoustofluidic device to fabricate heterotypic cell spheroids with complexity over cell architectures and components. By varying the concns. of cell suspension, we can precisely control the size of spheroids aggregated by a contact-free acoustic radiation force. By tuning the cell components including tumor cells, fibroblasts, and endothelial cells, heterotypic spheroids were controllably fabricated. These heterotypic spheroids can be used as a proof-of concept to model the spatial organization of tumor tissues. We demonstrated that the assembled components can self-assemble into layered structures as instructed by their cadherin expression. Finally, we demonstrated the acoustic assembly of mouse mammary gland components into spheroids and obsd. their maturation in culture. To conclude, we developed an acoustofluidic platform to fabricate complex spheroids with multiple components. We envision that this platform will pave the way for the high accuracy of spheroid fabrication and offer broad applications in numerous areas, such as tumor research, tissue engineering, developmental biol., and drug discovery.
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  116. 116
    Sebastian, A.; Buckle, A. M.; Markx, G. H. Formation of Multilayer Aggregates of Mammalian Cells by Dielectrophoresis. J. Micromechanics Microengineering 2006, 16, 1769,  DOI: 10.1088/0960-1317/16/9/003
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    Yasukawa, T.; Morishima, A.; Suzuki, M.; Yoshioka, J.; Yoshimoto, K.; Mizutani, F. Rapid Formation of Aggregates with Uniform Numbers of Cells Based on Three-Dimensional Dielectrophoresis. Anal. Sci. 2019, 35, 895,  DOI: 10.2116/analsci.19P074
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    117
    Rapid formation of aggregates with uniform numbers of cells based on three-dimensional dielectrophoresis
    Yasukawa, Tomoyuki; Ma, Asa Mouisin; Suzuki, Masato; Yoshioka, Junya; Yoshimoto, Keitaro; Mizutani, Fumio
    Analytical Sciences (2019), 35 (8), 895-901CODEN: ANSCEN; ISSN:0910-6340. (Japan Society for Analytical Chemistry)
    We applied a fabrication method for the formation of island organization of cells based on a three-dimensional (3D) device for neg. dielectrophoresis (n-DEP) to produce cell aggregates with uniform nos. of cells rapidly and simply. The intersections formed by rotating the interdigitated array (IDA) with two combs of hand electrodes on the upper substrate by 90° relative to the IDA with two combs on the lower substrate were prepd. in the device. The AC voltage was applied to a comb on the upper substrate and a comb on the lower substrate, while AC voltage with opposite phase was applied to another comb on the upper substrate and another comb on the lower substrate. Cells dispersed randomly were directed toward the intersections with relatively lower elec. fields due to n-DEP, which formed by AC voltage applied bands with the identical phase, resulting in the formation of island patterns of cells. The cells accumulated at intersections were promoted to form the cell aggregates due to the close contact together. The prodn. of cell aggregations adhered together was easily found by the dispersion behavior after switching the applied frequency to convert the cellular pattern. When cells were accumulated at the intersections by n-DEP for 45 min, almost accumulations of cells were adhered together, and hence a formations of cell aggregations. By using the present method, we can rapidly and simply fabricate cell aggregations with a uniform no. of cells.
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  118. 118
    Chong, D. T.; Liu, X. S.; Ma, H. J.; Huang, G. Y.; Han, Y. L.; Cui, X. Y.; Yan, J. J.; Xu, F. Advances in Fabricating Double-Emulsion Droplets and Their Biomedical Applications. Microfluidics and Nanofluidics. 2015, 19, 1071,  DOI: 10.1007/s10404-015-1635-8
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    118
    Advances in fabricating double-emulsion droplets and their biomedical applications
    Chong, Dao Tong; Liu, Xin Shi; Ma, Hua Jie; Huang, Guo You; Han, Yu Long; Cui, Xing Ye; Yan, Jun Jie; Xu, Feng
    Microfluidics and Nanofluidics (2015), 19 (5), 1071-1090CODEN: MNIAAR; ISSN:1613-4982. (Springer)
    Double-emulsion droplets have found widespread applications in various engineering and biomedical fields because of their capability in encapsulating different components in each layer. The conventional double-emulsion method is the two-stage stirring emulsification method, which suffers from poor monodispersity and low encapsulation efficiency. With recent advances in microfabrication, some novel methods for fabricating double-emulsion droplets have been developed, including microfluidic emulsification (double-T-junction microchannel, double-cross-shaped microchannel and several three-dimensional microchannels), membrane emulsification and coaxial electrospraying. These methods have shown significantly improved droplet features (e.g., size, size uniformity, thickness of each layer, generation throughput capability). Herein, this paper first reviews the state-of-art approaches for fabricating double-emulsion droplets and discusses their advantages and disadvantages. The applications of double-emulsion droplets in biomedical fields, including cell encapsulation, drug delivery and controlled release, and synthetic biol. are also discussed. In conclusion, future perspectives are given.
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  119. 119
    Qu, F.; Zhao, S.; Cheng, G.; Rahman, H.; Xiao, Q.; Chan, R. W. Y.; Ho, Y. P. Double Emulsion-Pretreated Microwell Culture for the in Vitro Production of Multicellular Spheroids and Their in Situ Analysis. Microsystems Nanoeng. 2021,  DOI: 10.1038/s41378-021-00267-w
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    Zhan, Z.; Liu, Z.; Nan, H.; Li, J.; Xie, Y.; Hu, C. Heterogeneous Spheroids with Tunable Interior Morphologies by Droplet-Based Microfluidics. Biofabrication 2022, 14 (2), 025024,  DOI: 10.1088/1758-5090/ac5e12
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    Sharma, S.; Srisa-Art, M.; Scott, S.; Asthana, A.; Cass, A. Droplet-Based Micro Fluidics. Methods Mol. Biol. 2013, 949, 207,  DOI: 10.1007/978-1-62703-134-9_15
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    Droplet-based microfluidics
    Sharma, Sanjiv; Srisa-Art, Monpichar; Scott, Steven; Asthana, Amit; Cass, Anthony
    Methods in Molecular Biology (New York, NY, United States) (2013), 949 (Microfluidic Diagnostics), 207-230CODEN: MMBIED; ISSN:1064-3745. (Springer)
    A review. Drop let-based microfluidics or digital microfluidics is a subclass of microfluidic devices, wherein droplets are generated using active or passive methods. The active method for generation of droplets involves the use of an external factor such as an elec. field for droplet generation. Two techniques that fall in this category are dielectrophoresis (DEP) and electrowetting on dielec. (EWOD). In passive methods, the droplet generation depends on the geometry and dimensions of the device. T-junction and flow focusing methods are examples of passive methods used for generation of droplets. In this chapter the methods used for droplet generation, mixing of contents of droplets, and the manipulation of droplets are described in brief. A review of the applications of digital microfluidics with emphasis on the last decade is presented.
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  122. 122
    Sartipzadeh, O.; Naghib, S. M.; Seyfoori, A.; Rahmanian, M.; Fateminia, F. S. Controllable Size and Form of Droplets in Microfluidic-Assisted Devices: Effects of Channel Geometry and Fluid Velocity on Droplet Size. Mater. Sci. Eng., C 2020, 109, 110606,  DOI: 10.1016/j.msec.2019.110606
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    122
    Controllable size and form of droplets in microfluidic-assisted devices: Effects of channel geometry and fluid velocity on droplet size
    Sartipzadeh, Omid; Naghib, Seyed Morteza; Seyfoori, Amir; Rahmanian, Mehdi; Fateminia, Fatemeh Sadat
    Materials Science & Engineering, C: Materials for Biological Applications (2020), 109 (), 110606CODEN: MSCEEE; ISSN:0928-4931. (Elsevier B.V.)
    Droplet-based microfluidic assisted devices have proposed an extensive interest in many applications such as lab-on-a-chip technologies as well as chem./biol./nanomaterial prepn., chem. engineering, drug delivery, tissue engineering and biosensing. Here, a computational fluid dynamic model was developed for deep understanding of the droplet size and formation in a flow-focusing (FF) microchannel with consideration of the continuous phase (non-Newtonian fluid). The simulations presented an alternative method to achieve insights into this complicated process. In the following for the first time, the role of channel geometry, channel aspect ratio and flow rate ratio on droplet features including the mechanism of droplet formation, diam./vol. of droplet, velocity/amt. of droplet formation, and final shape/size of the generated droplets were fully described. These findings could remarkably derive desirable protocols to control droplets characteristics comprising their size and shape in non-Newtonian fluids. Moreover, level set (LS) method was used for scrutinizing the droplet-breaking procedure in the microfluidic FF devices. The results showed that different droplet sizes could be prepd. with changing the various parameters, demonstrating many challenges in various applications including lab-on-a-chip, cell encapsulation, drug delivery, tissue engineering, biosensing and bioimaging could be successfully addressed.
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  123. 123
    Panhwar, M. H.; Czerwinski, F.; Dabbiru, V. A. S.; Komaragiri, Y.; Fregin, B.; Biedenweg, D.; Nestler, P.; Pires, R. H.; Otto, O. High-Throughput Cell and Spheroid Mechanics in Virtual Fluidic Channels. Nat. Commun. 2020,  DOI: 10.1038/s41467-020-15813-9
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    Marín, A. G.; Campo-Cortés, F.; Gordillo, J. M. Generation of Micron-Sized Drops and Bubbles through Viscous Coflows. Colloids Surfaces A Physicochem. Eng. Asp. 2009, 344, 2,  DOI: 10.1016/j.colsurfa.2008.09.033
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    Lashkaripour, A.; Rodriguez, C.; Mehdipour, N.; Mardian, R.; McIntyre, D.; Ortiz, L.; Campbell, J.; Densmore, D. Machine Learning Enables Design Automation of Microfluidic Flow-Focusing Droplet Generation. Nat. Commun. 2021,  DOI: 10.1038/s41467-020-20284-z
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    Lee, D.; Cha, C. The Combined Effects of Co-Culture and Substrate Mechanics on 3d Tumor Spheroid Formation within Microgels Prepared via Flow-Focusing Microfluidic Fabrication. Pharmaceutics 2018, 10, 229,  DOI: 10.3390/pharmaceutics10040229
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    126
    The combined effects of co-culture and substrate mechanics on 3D tumor spheroid formation within microgels prepared via flow-focusing microfluidic fabrication
    Lee, Dongjin; Cha, Chaenyung
    Pharmaceutics (2018), 10 (4), 229CODEN: PHARK5; ISSN:1999-4923. (MDPI AG)
    Tumor spheroids are considered a valuable three dimensional (3D) tissue model to study various aspects of tumor physiol. for biomedical applications such as tissue engineering and drug screening as well as basic scientific endeavors, as several cell types can efficiently form spheroids by themselves in both suspension and adherent cell cultures. However, it is more desirable to utilize a 3D scaffold with tunable properties to create more physiol. relevant tumor spheroids as well as optimize their formation. In this study, bioactive spherical microgels supporting 3D cell culture are fabricated by a flow-focusing microfluidic device. Uniform-sized aq. droplets of gel precursor soln. dispersed with cells generated by the microfluidic device are photocrosslinked to fabricate cell-laden microgels. Their mech. properties are controlled by the concn. of gel-forming polymer. Using breast adenocarcinoma cells, MCF-7, the effect of mech. properties of microgels on their proliferation and the eventual spheroid formation was explored. Furthermore, the tumor cells are co-cultured with macrophages of fibroblasts, which are known to play a prominent role in tumor physiol., within the microgels to explore their role in spheroid formation. Taken together, the results from this study provide the design strategy for creating tumor spheroids utilizing mech.-tunable microgels as 3D cell culture platform.
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  127. 127
    Choi, K.; Ng, A. H. C.; Fobel, R.; Wheeler, A. R. Digital Microfluidics. Annual Review of Analytical Chemistry. 2012, 5, 413,  DOI: 10.1146/annurev-anchem-062011-143028
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    Digital microfluidics
    Choi, Kihwan; Ng, Alphonsus H. C.; Fobel, Ryan; Wheeler, Aaron R.
    Annual Review of Analytical Chemistry (2012), 5 (), 413-440CODEN: ARACFU; ISSN:1936-1327. (Annual Reviews Inc.)
    A review. Digital microfluidics (DMF) is an emerging liq.-handling technol. that enables individual control over droplets on an open array of electrodes. These picoliter- to microliter-sized droplets, each serving as an isolated vessel for chem. processes, can be made to move, merge, split, and dispense from reservoirs. Because of its unique advantages, including simple instrumentation, flexible device geometry, and easy coupling with other technologies, DMF is being applied to a wide range of fields. In this review, we summarize the state of the art of DMF technol. from the perspective of anal. chem. in sections describing the theory of droplet actuation, device fabrication and integration, and applications.
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    Hong, J.; Kim, Y. K.; Won, D. J.; Kim, J.; Lee, S. J. Three-Dimensional Digital Microfluidic Manipulation of Droplets in Oil Medium. Sci. Rep. 2015,  DOI: 10.1038/srep10685
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    Kothamachu, V. B.; Zaini, S.; Muffatto, F. Role of Digital Microfluidics in Enabling Access to Laboratory Automation and Making Biology Programmable. SLAS Technology. 2020, 25, 411,  DOI: 10.1177/2472630320931794
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    129
    Role of digital microfluidics in enabling access to laboratory automation and making biology programmable
    Kothamachu, Varun B.; Zaini, Sabrina; Muffatto, Federico
    SLAS Technology (2020), 25 (5), 411-426CODEN: STLECS; ISSN:2472-6303. (Sage Publications)
    A review. Digital microfluidics (DMF) is a liq. handling technique that has been demonstrated to automate biol. experimentation in a low-cost, rapid, and programmable manner. This review discusses the role ofDMF as a "digital bioconverter"-a tool to connect the digital aspects ofthe design-build-learn cycle with the phys. execution ofexperiments. Several applications are reviewed to demonstrate the utility of DMF as a digital bioconverter, namely, genetic engineering, sample prepn. for sequencing and mass spectrometry, and enzyme-, immuno-, and cell-based screening assays. These applications show that DMF has great potential in the role ofa centralized execution platform in a fully integrated pipeline for the prodn. of novel organisms and biomols. In this paper, we discuss how the function of a DMF device within such a pipeline is highly dependent on integration with different sensing techniques and methodologies from machine learning and big data. In addn. to that, we examine how the capacity of DMF can in some cases be limited by known tech. and operational challenges and how consolidated efforts in overcoming these challenges will be key to the development of DMF as a major enabling technol. in the computer-aided biol. framework.
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    Hwang, Y. S.; Kim, J.; Yoon, H. J.; Kang, J. I.; Park, K. H.; Bae, H. Microwell-Mediated Cell Spheroid Formation and Its Applications. Macromolecular Research. 2018, 26, 1,  DOI: 10.1007/s13233-018-6002-7
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    130
    Microwell-mediated cell spheroid formation and its applications
    Hwang, Yu-Shik; Kim, Jinseok; Yoon, Hee Jeong; Kang, Ji In; Park, Ki-Ho; Bae, Hojae
    Macromolecular Research (2018), 26 (1), 1-8CODEN: MRAECT; ISSN:1598-5032. (Polymer Society of Korea)
    A review. There is a continuing attempt to study cell to cell interactions to control the growth, function, and differentiation of cells. One approach that can be used to assess representative cell to cell interactions is by using three-dimensional cell spheroids, also referred to as cell aggregates, generated by adhering cells to each other through cell-adhesion mols. such as cadherin. Compared with the conventional two-dimensional cell monolayer, the cell spheroid offers a more realistic platform to predict cell behavior in high-throughput manner. To recapitulate the cell spheroid formation in vitro, microwell-mediated culture system has become a robust and efficient tool for providing uniform-sized spheroids. In this review, the authors first iterate the recent developments and innovations in microwell-mediated cell culture platform, focusing on formation and function of cell spheroids using various cells such as embryonic stem cells, postnatal stem cells, and somatic cells. Furthermore, the recent advancements in applications of cell spheroids generated from microwell-mediated culture system is covered. The discussion on the integrative biol. regarding cell to cell interaction and other biol. events in cell spheroid is another focal point of this review.
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  131. 131
    Kim, D.; Kim, K.; Park, J. Y. Novel Microwell with a Roof Capable of Buoyant Spheroid Culture. Lab Chip 2021, 21, 1974,  DOI: 10.1039/D0LC01295E
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    131
    Novel microwell with a roof capable of buoyant spheroid culture
    Kim, Daehan; Kim, Kideok; Park, Joong Yull
    Lab on a Chip (2021), 21 (10), 1974-1986CODEN: LCAHAM; ISSN:1473-0189. (Royal Society of Chemistry)
    Microwells are used in studies to mimic the in vivo environment through an in vitro environment by generating three-dimensional cell spheroids. These microwells have been fabricated in various shapes using different methods according to the research purpose. However, because all microwells up to now have an open top, it has been difficult to culture spheroids of floating cells due to their low d., such as human adipose-derived stem cells (hASCs) that differentiate into adipocytes. Therefore, the labor-intensive hanging droplet method has been mainly used for the study of adipocytes. Here, we introduce a sigma-well, which is a microwell in the shape of the Greek letter sigma (σ) with a roof. Because of its unique shape, the sigma-well is advantageous for the culture of floating cells by reducing cell loss and external interference. The sigma-well was fabricated using the principle of surface tension of polydimethylsiloxane as well as air trapping and thermal expansion. Unlike conventional microwells, because the center of the bottom surface and the inlet of the sigma-well are not located on the same line and have a difference of approx. 218μm, the spheroids are cultured more stably and may not escape the cavity. In this study, hASC and adipocyte spheroids differentiated using these sigma-wells were successfully cultured. In addn., through cytokine diffusion simulation, it was confirmed that the diffusion and mass transfer in the sigma-well was lower than that in the conventional microwell. It is expected that the morphol. features of the sigma-well, which cannot be easily obtained by other methods, can be beneficial for the study of buoyant cell types such as adipocytes.
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    Charnley, M.; Textor, M.; Khademhosseini, A.; Lutolf, M. P. Integration Column: Microwell Arrays for Mammalian Cell Culture. Integr. Biol. 2009, 1, 625,  DOI: 10.1039/b918172p
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    132
    Integration column: microwell arrays for mammalian cell culture
    Charnley, Mirren; Textor, Marcus; Khademhosseini, Ali; Lutolf, Matthias P.
    Integrative Biology (2009), 1 (11-12), 625-634CODEN: IBNIFL ISSN:. (Royal Society of Chemistry)
    A review. Microwell arrays have emerged as robust and versatile alternatives to conventional mammalian cell culture substrates. Using std. microfabrication processes, biomaterials surfaces can be topog. patterned to comprise high-d. arrays of micron-sized cavities with desirable geometry. Hundreds to thousands of individual cells or cell colonies with controlled size and shape can be trapped in these cavities by simple gravitational sedimentation. Efficient long-term cell confinement allows for parallel analyses and manipulation of cell fate during in vitro culture. These live-cell arrays have already found applications in cell biol., for example to probe the effect of cell colony size on embryonic stem cell differentiation, to dissect the heterogeneity in single cell proliferation kinetics of neural or hematopoietic stem/progenitor cell populations, or to elucidate the role of cell shape on cell function. Here, we highlight the key applications of these platforms, hopefully inspiring biologists to apply these systems for their own studies.
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  133. 133
    Pedde, R. D.; Mirani, B.; Navaei, A.; Styan, T.; Wong, S.; Mehrali, M.; Thakur, A.; Mohtaram, N. K.; Bayati, A.; Dolatshahi-Pirouz, A.; Nikkhah, M.; Willerth, S. M.; Akbari, M. Emerging Biofabrication Strategies for Engineering Complex Tissue Constructs. Adv. Mater. 2017, 29, 1606061,  DOI: 10.1002/adma.201606061
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    Manzoor, A. A.; Romita, L.; Hwang, D. K. A Review on Microwell and Microfluidic Geometric Array Fabrication Techniques and Its Potential Applications in Cellular Studies. Can. J. Chem. Eng. 2021, 99, 61,  DOI: 10.1002/cjce.23875
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    134
    A review on microwell and microfluidic geometric array fabrication techniques and its potential applications in cellular studies
    Manzoor, Ahmad Ali; Romita, Lauren; Hwang, Dae Kun
    Canadian Journal of Chemical Engineering (2021), 99 (1), 61-96CODEN: CJCEA7; ISSN:0008-4034. (John Wiley & Sons, Inc.)
    The ability to trap precise quantities of cells or particles into confined areas has numerous applications for biol. purposes. In particular, single cell trapping has received considerable attention because it permits the study of heterogeneity in a population, while trapping larger groups of cells have been used to form aggregates. Among several methods, the use of microwell offers a simple platform to capture cells or particles using hydrodynamic forces. This review examines the use of microwells in both static and microfluidic environments, and the application of microfluidic geometric arrays for trapping. This paper discusses the design and fabrication methods of microwells and compares the trapping and release techniques used in both static and microfluidics-integrated microwells. Finally, we will summarize novel microfluidic geometric arrays used to capture cells or particles through hydrodynamic trapping.
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  135. 135
    Rousset, N.; Monet, F.; Gervais, T. Simulation-Assisted Design of Microfluidic Sample Traps for Optimal Trapping and Culture of Non-Adherent Single Cells, Tissues, and Spheroids. Sci. Rep. 2017,  DOI: 10.1038/s41598-017-00229-1
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    Rismani Yazdi, S.; Shadmani, A.; Bürgel, S. C.; Misun, P. M.; Hierlemann, A.; Frey, O. Adding the “heart” to Hanging Drop Networks for Microphysiological Multi-Tissue Experiments. Lab Chip 2015, 15, 4138,  DOI: 10.1039/C5LC01000D
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    136
    Adding the 'heart' to hanging drop networks for microphysiological multi-tissue experiments
    Rismani Yazdi, Saeed; Shadmani, Amir; Burgel, Sebastian C.; Misun, Patrick M.; Hierlemann, Andreas; Frey, Olivier
    Lab on a Chip (2015), 15 (21), 4138-4147CODEN: LCAHAM; ISSN:1473-0189. (Royal Society of Chemistry)
    Microfluidic hanging-drop networks enable culturing and anal. of 3D microtissue spheroids derived from different cell types under controlled perfusion and investigating inter-tissue communication in multi-tissue formats. In this paper we introduce a compact on-chip pumping approach for flow control in hanging-drop networks. The pump includes one pneumatic chamber located directly above one of the hanging drops and uses the surface tension at the liq.-air-interface for flow actuation. Control of the pneumatic protocol provides a wide range of unidirectional pulsatile and continuous flow profiles. With the proposed concept several independent hanging-drop networks can be operated in parallel with only one single pneumatic actuation line at high fidelity. Closed-loop medium circulation between different organ models for multi-tissue formats and multiple simultaneous assays in parallel are possible. Finally, we implemented a real-time feedback control-loop of the pump actuation based on the beating of a human iPS-derived cardiac microtissue cultured in the same system. This configuration allows for simulating physiol. effects on the heart and their impact on flow circulation between the organ models on chip.
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  137. 137
    Suryaprakash, R. T. C.; Kujan, O.; Shearston, K.; Farah, C. S. Three-Dimensional Cell Culture Models to Investigate Oral Carcinogenesis: A Scoping Review. International Journal of Molecular Sciences. 2020, 21, 9520,  DOI: 10.3390/ijms21249520
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    137
    Three-dimensional cell culture models toinvestigate oral carcinogenesis: a scoping review
    Suryaprakash, Ravi Teja Chitturi; Kujan, Omar; Shearston, Kate; Farah, Camile S.
    International Journal of Molecular Sciences (2020), 21 (24), 9520CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)
    A review. Three-dimensional (3-D) cell culture models, such as spheroids, organoids, and organotypic cultures, are more physiol. representative of the human tumor microenvironment (TME) than traditional two-dimensional (2-D) cell culture models. They have been used as in vitro models to investigate various aspects of oral cancer but, to date, have not be widely used in investigations of the process of oral carcinogenesis. The aim of this scoping review was to evaluate the use of 3-D cell cultures in oral squamous cell carcinoma (OSCC) research, with a particular emphasis on oral carcinogenesis studies. Databases (PubMed, Scopus, and Web of Science) were systematically searched to identify research applying 3-D cell culture techniques to cells from normal, dysplastic, and malignant oral mucosae. A total of 119 studies were included for qual. anal. including 53 studies utilizing spheroids, 62 utilizing organotypic cultures, and 4 using organoids. We found that 3-D oral carcinogenesis studies had been limited to just two organotypic culture models and that to date, spheroids and organoids had not been utilized for this purpose. Spheroid culture was most frequently used as a tumorosphere forming assay and the organoids cultured from human OSCCs most often used in drug sensitivity testing. These results indicate that there are significant opportunities to utilize 3-D cell culture to explore the development of oral cancer, particularly as the physiol. relevance of these models continues to improve.
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  138. 138
    Shen, H.; Cai, S.; Wu, C.; Yang, W.; Yu, H.; Liu, L. Recent Advances in Three-Dimensional Multicellular Spheroid Culture and Future Development. Micromachines. 2021, 12, 96,  DOI: 10.3390/mi12010096
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    Barisam, M.; Saidi, M. S.; Kashaninejad, N.; Vadivelu, R.; Nguyen, N. T. Numerical Simulation of the Behavior of Toroidal and Spheroidal Multicellular Aggregates in Microfluidic Devices with Microwell and U-Shaped Barrier. Micromachines 2017, 8, 358,  DOI: 10.3390/mi8120358
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    Behroodi, E.; Latifi, H.; Bagheri, Z.; Ermis, E.; Roshani, S.; Salehi Moghaddam, M. A Combined 3D Printing/CNC Micro-Milling Method to Fabricate a Large-Scale Microfluidic Device with the Small Size 3D Architectures: An Application for Tumor Spheroid Production. Sci. Rep. 2020,  DOI: 10.1038/s41598-020-79015-5
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    Torisawa, Y. S.; Mosadegh, B.; Luker, G. D.; Morell, M.; O’Shea, K. S.; Takayama, S. Microfluidic Hydrodynamic Cellular Patterning for Systematic Formation of Co-Culture Spheroids. Integr. Biol. 2009, 1, 649,  DOI: 10.1039/b915965g
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    141
    Microfluidic hydrodynamic cellular patterning for systematic formation of co-culture spheroids
    Torisawa, Yu-suke; Mosadegh, Bobak; Luker, Gary D.; Morell, Maria; O'Shea, K. Sue; Takayama, Shuichi
    Integrative Biology (2009), 1 (11-12), 649-654CODEN: IBNIFL ISSN:. (Royal Society of Chemistry)
    This paper describes a microfluidic method to form co-culture spheroids of various geometries and compns. in order to manipulate cell-cell interaction dynamics. The cellular patterning is performed in a two-layered microfluidic device that sandwiches a semi-porous membrane so that flow occurs from the top channel through the membrane to the bottom channel. Arbitrary cellular arrangements are enabled by regulating the geometric features of the bottom channel so that as culture media drains, the flow hydrodynamically focuses (aggregates) cells onto the membrane only over the regions of the bottom channel. Furthermore, when the top channel has multiple inlets, cells can be seeded in adjacent laminar streams, allowing different cell types to be patterned simultaneously in well defined spatial arrangements. Interestingly, the initial cell positioning of certain cell types can result in two juxtaposed non-concentric "Janus" spheroids, rather than homogeneous mixts. or layered shell structures. Therefore, the initial position of cells prior to aggregation can influence the final configuration within a co-culture spheroid. When Janus spheroids were constructed from mouse embryonic stem (mES) cells and hepatocytes, the mES cells differentiated in a spatially distinct pattern dictated by the position of the hepatocytes. This contrasts with uniform mES differentiation obsd. when co-culture spheroids are formed by the conventional method of randomly mixing the two cell types. This cellular patterning method opens new possibilities for understanding and manipulating interactions between different cell types in 3D.
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  142. 142
    Wang, T.; Green, R.; Nair, R. R.; Howell, M.; Mohapatra, S.; Guldiken, R.; Mohapatra, S. S. Surface Acoustic Waves (SAW)-Based Biosensing for Quantification of Cell Growth in 2D and 3D Cultures. Sensors (Switzerland) 2015, 15, 32045,  DOI: 10.3390/s151229909
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    142
    Surface Acoustic Waves (SAW)-Based Biosensing for Quantification of Cell Growth in 2D and 3D Cultures
    Wang Tao; Green Ryan; Nair Rajesh Ramakrishnan; Howell Mark; Mohapatra Subhra; Guldiken Rasim; Mohapatra Shyam Sundar; Wang Tao; Guldiken Rasim; Green Ryan; Howell Mark; Mohapatra Subhra; Nair Rajesh Ramakrishnan; Mohapatra Shyam Sundar; Nair Rajesh Ramakrishnan
    Sensors (Basel, Switzerland) (2015), 15 (12), 32045-55 ISSN:.
    Detection and quantification of cell viability and growth in two-dimensional (2D) and three-dimensional (3D) cell cultures commonly involve harvesting of cells and therefore requires a parallel set-up of several replicates for time-lapse or dose-response studies. Thus, developing a non-invasive and touch-free detection of cell growth in longitudinal studies of 3D tumor spheroid cultures or of stem cell regeneration remains a major unmet need. Since surface acoustic waves (SAWs) permit mass loading-based biosensing and have been touted due to their many advantages including low cost, small size and ease of assembly, we examined the potential of SAW-biosensing to detect and quantify cell growth. Herein, we demonstrate that a shear horizontal-surface acoustic waves (SH-SAW) device comprising two pairs of resonators consisting of interdigital transducers and reflecting fingers can be used to quantify mass loading by the cells in suspension as well as within a 3D cell culture platform. A 3D COMSOL model was built to simulate the mass loading response of increasing concentrations of cells in suspension in the polydimethylsiloxane (PDMS) well in order to predict the characteristics and optimize the design of the SH-SAW biosensor. The simulated relative frequency shift from the two oscillatory circuit systems (one of which functions as control) were found to be concordant to experimental data generated with RAW264.7 macrophage and A549 cancer cells. In addition, results showed that SAW measurements per se did not affect viability of cells. Further, SH-SAW biosensing was applied to A549 cells cultured on a 3D electrospun nanofiber scaffold that generate tumor spheroids (tumoroids) and the results showed the device's ability to detect changes in tumor spheroid growth over the course of eight days. Taken together, these results demonstrate the use of SH-SAW device for detection and quantification of cell growth changes over time in 2D suspension cultures and in 3D cell culture models, which may have potential applications in both longitudinal 3D cell cultures in cancer biology and in regenerative medicine.
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  143. 143
    Abdallat, R. G.; Ahmad Tajuddin, A. S.; Gould, D. H.; Hughes, M. P.; Fatoyinbo, H. O.; Labeed, F. H. Process Development for Cell Aggregate Arrays Encapsulated in a Synthetic Hydrogel Using Negative Dielectrophoresis. Electrophoresis 2013, 34, 1059,  DOI: 10.1002/elps.201200459
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    143
    Process development for cell aggregate arrays encapsulated in a synthetic hydrogel using negative dielectrophoresis
    Abdallat, Rula G.; Ahmad Tajuddin, Aziela S.; Gould, David H.; Hughes, Michael P.; Fatoyinbo, Henry O.; Labeed, Fatima H.
    Electrophoresis (2013), 34 (7), 1059-1067CODEN: ELCTDN; ISSN:0173-0835. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Spatial patterning of cells is of great importance in tissue engineering and biotechnol., enabling, for example the creation of bottom-up histoarchitectures of heterogeneous cells, or cell aggregates for in vitro high-throughput toxicol. and therapeutic studies within 3D microenvironments. In this paper, a single-step process for creating peelable and resilient hydrogels, encapsulating arrays of biol. cell aggregates formed by neg. DEP has been devised. The dielectrophoretic trapping within low-energy regions of the DEP-dot array reduces cell exposure to high field stresses while creating distinguishable, evenly spaced arrays of aggregates. In addn. to using an optimal combination of PEG diacrylate pre-polymer soln. concn. and a novel UV exposure mechanism, total processing time was reduced. With a continuous phase medium of PEG diacrylate at 15% vol./vol. concn., effective dielectrophoretic cell patterned arrays and photo-polymn. of the mixt. was achieved within a 4 min period. This unique single-step process was achieved using a 30 s UV exposure time frame within a dedicated, wide exposure area DEP light box system. To demonstrate the developed process, aggregates of yeast, human leukemic (K562) and HeLa cells were immobilized in an array format within the hydrogel. Relative cell viability for both cells within the hydrogels, after maintaining them in appropriate iso-osmotic media, over a week period was greater than 90%.
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  144. 144
    Petta, D.; Basoli, V.; Pellicciotta, D.; Tognato, R.; Barcik, J.; Arrigoni, C.; Bella, E. D.; Armiento, A. R.; Candrian, C.; Richards, R. G.; Alini, M.; Moretti, M.; Eglin, D.; Serra, T. Sound-Induced Morphogenesis of Multicellular Systems for Rapid Orchestration of Vascular Networks. 2021, 13, 015004,  DOI: 10.1088/1758-5090/abbb9c
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    Chen, P.; Guven, S.; Usta, O. B.; Yarmush, M. L.; Demirci, U. Biotunable Acoustic Node Assembly of Organoids.. Adv. Healthcare Mater. 2015, 4, 1937,  DOI: 10.1002/adhm.201500279
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    145
    Biotunable Acoustic Node Assembly of Organoids
    Chen, Pu; Gueven, Sinan; Usta, Osman Berk; Yarmush, Martin L.; Demirci, Utkan
    Advanced Healthcare Materials (2015), 4 (13), 1937-1943CODEN: AHMDBJ; ISSN:2192-2640. (Wiley-VCH Verlag GmbH & Co. KGaA)
    There is no expanded citation for this reference.
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  146. 146
    Guex, A. G.; Di Marzio, N.; Eglin, D.; Alini, M.; Serra, T. The Waves That Make the Pattern: A Review on Acoustic Manipulation in Biomedical Research. Materials Today Bio. 2021, 10, 100110,  DOI: 10.1016/j.mtbio.2021.100110
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    146
    The waves that make the pattern: a review on acoustic manipulation in biomedical research
    Guex, A. G.; Di Marzio, N.; Eglin, D.; Alini, M.; Serra, T.
    Materials Today Bio (2021), 10 (), 100110CODEN: MTBAC2; ISSN:2590-0064. (Elsevier Ltd.)
    Novel approaches, combining technol., biomaterial design, and cutting-edge cell culture, have been increasingly considered to advance the field of tissue engineering and regenerative medicine. Within this context, acoustic manipulation to remotely control spatial cellular organization within a carrier matrix has arisen as a particularly promising method during the last decade. Acoustic or sound-induced manipulation takes advantage of hydrodynamic forces exerted on systems of particles within a liq. medium by standing waves. Inorg. or org. particles, cells, or organoids assemble within the nodes of the standing wave, creating distinct patterns in response to the applied frequency and amplitude. Acoustic manipulation has advanced from micro- or nanoparticle arrangement in 2D to the assembly of multiple cell types or organoids into highly complex in vitro tissues. In this review, we discuss the past research achievements in the field of acoustic manipulation with particular emphasis on biomedical application. We survey microfluidic, open chamber, and high throughput devices for their applicability to arrange non-living and living units in buffer or hydrogels. We also investigate the challenges arising from different methods, and their prospects to gain a deeper understanding of in vitro tissue formation and application in the field of biomedical engineering.
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  147. 147
    Prasad, S.; Zhang, X.; Yang, M.; Ni, Y.; Parpura, V.; Ozkan, C. S.; Ozkan, M. Separation of Individual Neurons Using Dielectrophoretic Alternative Current Fields. J. Neurosci. Methods 2004, 135, 79,  DOI: 10.1016/j.jneumeth.2003.12.007
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    147
    Separation of individual neurons using dielectrophoretic alternative current fields
    Prasad Shalini; Zhang Xuan; Yang Mo; Ni Yingchun; Parpura Vladimir; Ozkan Cengiz S; Ozkan Mihrimah
    Journal of neuroscience methods (2004), 135 (1-2), 79-88 ISSN:0165-0270.
    Experimental investigations into the dynamics of neuronal networks are a fundamental step towards understanding how the nervous system works. Memory formation and development are associated with changes in the electrical activity of the neurons. To understand the changes in the electrical activity, it is essential to conduct in vitro studies on individual neurons. Hence, there is an enormous need to develop novel ways for isolating and localizing individual neurons. To this end, we designed and fabricated a 4x4 multiple microelectrode array system to spatially arrange neurons by generating dielectrophoretic traps using gradient alternating current (AC) fields. We characterized the electric field distribution inside our test platform by using three-dimensional finite element modeling (FEM) and estimated the location of neurons over the electrode array. As the first stage in forming a neuronal network, dielectrophoretic AC fields were employed to separate the neurons from the glial cells and to position individual neurons over single electrodes. The extracellular electrical activity from a single neuron was recorded. The frequency spectrum of the electrical activity was generated using fast Fourier transformation analysis (FFT) to determine the characteristic burst rates of individual neurons.
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  148. 148
    Schneider, S.; Gruner, D.; Richter, A.; Loskill, P. Membrane Integration into PDMS-Free Microfluidic Platforms for Organ-on-Chip and Analytical Chemistry Applications. Lab on a Chip. 2021, 21, 1866,  DOI: 10.1039/D1LC00188D
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    148
    Membrane integration into PDMS-free microfluidic platforms for organ-on-chip and analytical chemistry applications
    Schneider, Stefan; Gruner, Denise; Richter, Andreas; Loskill, Peter
    Lab on a Chip (2021), 21 (10), 1866-1885CODEN: LCAHAM; ISSN:1473-0189. (Royal Society of Chemistry)
    A review. Membranes play a crucial role in many microfluidic systems, enabling versatile applications in highly diverse research fields. However, the tight and robust integration of membranes into microfluidic systems requires complex fabrication processes. Most integration approaches, so far, rely on polydimethylsiloxane (PDMS) as base material for the microfluidic chips. Several limitations of PDMS have resulted in the transition of many microfluidic approaches to PDMS-free systems using alternative materials such as thermoplastics. To integrate membranes in those PDMS-free systems, novel alternative approaches are required. This review provides an introduction into microfluidic systems applying membrane technol. for anal. systems and organ-on-chip as well as a comprehensive overview of methods for the integration of membranes into PDMS-free systems. The overview and examples will provide a valuable resource and starting point for any researcher that is aiming at implementing membranes in microfluidic systems without using PDMS.
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  149. 149
    Azizgolshani, H.; Coppeta, J. R.; Vedula, E. M.; Marr, E. E.; Cain, B. P.; Luu, R. J.; Lech, M. P.; Kann, S. H.; Mulhern, T. J.; Tandon, V.; Tan, K.; Haroutunian, N. J.; Keegan, P.; Rogers, M.; Gard, A. L.; Baldwin, K. B.; de Souza, J. C.; Hoefler, B. C.; Bale, S. S.; Kratchman, L. B.; Zorn, A.; Patterson, A.; Kim, E. S.; Petrie, T. A.; Wiellette, E. L.; Williams, C.; Isenberg, B. C.; Charest, J. L. High-Throughput Organ-on-Chip Platform with Integrated Programmable Fluid Flow and Real-Time Sensing for Complex Tissue Models in Drug Development Workflows. Lab Chip 2021, 21, 1454,  DOI: 10.1039/D1LC00067E
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    149
    High-throughput organ-on-chip platform with integrated programmable fluid flow and real-time sensing for complex tissue models in drug development workflows
    Azizgolshani, H.; Coppeta, J. R.; Vedula, E. M.; Marr, E. E.; Cain, B. P.; Luu, R. J.; Lech, M. P.; Kann, S. H.; Mulhern, T. J.; Tandon, V.; Tan, K.; Haroutunian, N. J.; Keegan, P.; Rogers, M.; Gard, A. L.; Baldwin, K. B.; de Souza, J. C.; Hoefler, B. C.; Bale, S. S.; Kratchman, L. B.; Zorn, A.; Patterson, A.; Kim, E. S.; Petrie, T. A.; Wiellette, E. L.; Williams, C.; Isenberg, B. C.; Charest, J. L.
    Lab on a Chip (2021), 21 (8), 1454-1474CODEN: LCAHAM; ISSN:1473-0189. (Royal Society of Chemistry)
    Drug development suffers from a lack of predictive and human-relevant in vitro models. Organ-on-chip (OOC) technol. provides advanced culture capabilities to generate physiol. appropriate, human-based tissue in vitro, therefore providing a route to a predictive in vitro model. However, OOC technologies are often created at the expense of throughput, industry-std. form factors, and compatibility with state-of-the-art data collection tools. Here we present an OOC platform with advanced culture capabilities supporting a variety of human tissue models including liver, vascular, gastrointestinal, and kidney. The platform has 96 devices per industry std. plate and compatibility with contemporary high-throughput data collection tools. Specifically, we demonstrate programmable flow control over two physiol. relevant flow regimes: perfusion flow that enhances hepatic tissue function and high-shear stress flow that aligns endothelial monolayers. In addn., we integrate elec. sensors, demonstrating quantification of barrier function of primary gut colon tissue in real-time. We utilize optical access to the tissues to directly quantify renal active transport and oxygen consumption via integrated oxygen sensors. Finally, we leverage the compatibility and throughput of the platform to screen all 96 devices using high content screening (HCS) and evaluate gene expression using RNA sequencing (RNA-seq). By combining these capabilities in one platform, physiol.-relevant tissues can be generated and measured, accelerating optimization of an in vitro model, and ultimately increasing predictive accuracy of in vitro drug screening.
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  150. 150
    Essaouiba, A.; Jellali, R.; Shinohara, M.; Scheidecker, B.; Legallais, C.; Sakai, Y.; Leclerc, E. Analysis of the Behavior of 2D Monolayers and 3D Spheroid Human Pancreatic Beta Cells Derived from Induced Pluripotent Stem Cells in a Microfluidic Environment. J. Biotechnol. 2021, 330, 45,  DOI: 10.1016/j.jbiotec.2021.02.009
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    150
    Analysis of the behavior of 2D monolayers and 3D spheroid human pancreatic beta cells derived from induced pluripotent stem cells in a microfluidic environment
    Essaouiba, Amal; Jellali, Rachid; Shinohara, Marie; Scheidecker, Benedikt; Legallais, Cecile; Sakai, Yasuyuki; Leclerc, Eric
    Journal of Biotechnology (2021), 330 (), 45-56CODEN: JBITD4; ISSN:0168-1656. (Elsevier B.V.)
    The limited availability of primary human β-cells/islets and their quality (due to donor diversity) restrict the development of in vitro models for diabetes research. Human induced pluripotent stem cells (hiPSCs) may be a promising cell-source for diabetes studies, anti-diabetic drug screening and personalized therapies. However, achieving levels of maturity/functionality that are comparable to the in vivo situation and islets rebuilt from iPSCs is still challenging. Here, we compare and discuss two strategies for culturing human pancreatic β-cells derived from hiPSCs in microfluidic biochips. First, we confirmed that the protocol in conventional Petri 2D monolayer led to insulin, PDX1 and MAFA pos. staining, to C-Peptide productive cells, and to tissue responsive to high/low glucose and GLP1 stimulation. This protocol and its subsequent modifications (including extracellular matrix coating, cell adhesion time, cell inoculation d., flow rate) was not successful in the 2D biochip culture. We proposed a second strategy using 3D spheroids created from honeycomb static cultures. Spheroids in static expts. carried out over 14 days demonstrated that they expressed high levels of β-cell markers (INS mRNA) and higher α-cell markers (GCG mRNA and glucagon pos. staining), when compared to Petri 2D cultures. Furthermore, the 3D spheroids were specifically able to secrete insulin in response to both high/low glucose stimulation and GLP1 exposure. The spheroids were successfully inoculated into biochips and maintained for 10 days in perfusion. The 3D biochip cultures increased mRNA levels of GCG and maintained high levels of β-cell markers and responsiveness to both high/low glucose and GLP1 stimulation. Finally, C-peptide and insulin secretion were higher in biochips when compared to static spheroids. These results illustrate the promising potential for hiPSCs derived β-cells and their spheroid-based pancreas-on-chip model for pancreatic disease/diabetes modeling and anti-diabetic drug screening.
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  151. 151
    Bovard, D.; Iskandar, A.; Luettich, K.; Hoeng, J.; Peitsch, M. C. Organs-on-a-Chip. Toxicol. Res. Appl. 2017, 1, 239784731772635,  DOI: 10.1177/2397847317726351
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    Lancaster, M. A.; Huch, M. Disease Modelling in Human Organoids. DMM Dis. Model. Mech. 2019,  DOI: 10.1242/dmm.039347
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    Benam, K. H.; Dauth, S.; Hassell, B.; Herland, A.; Jain, A.; Jang, K. J.; Karalis, K.; Kim, H. J.; MacQueen, L.; Mahmoodian, R.; Musah, S.; Torisawa, Y. S.; Van Der Meer, A. D.; Villenave, R.; Yadid, M.; Parker, K. K.; Ingber, D. E. Engineered in Vitro Disease Models. Annu. Rev. Pathol. Mech. Dis. 2015, 10, 195,  DOI: 10.1146/annurev-pathol-012414-040418
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    Engineered In Vitro Disease Models
    Benam, Kambez H.; Dauth, Stephanie; Hassell, Bryan; Herland, Anna; Jain, Abhishek; Jang, Kyung-Jin; Karalis, Katia; Kim, Hyun Jung; MacQueen, Luke; Mahmoodian, Roza; Musah, Samira; Torisawa, Yu-suke; van der Meer, Andries D.; Villenave, Remi; Yadid, Moran; Parker, Kevin K.; Ingber, Donald E.
    Annual Review of Pathology: Mechanisms of Disease (2015), 10 (), 195-262CODEN: ARPMCU; ISSN:1553-4006. (Annual Reviews)
    A review. The ultimate goal of most biomedical research is to gain greater insight into mechanisms of human disease or to develop new and improved therapies or diagnostics. Although great advances have been made in terms of developing disease models in animals, such as transgenic mice, many of these models fail to faithfully recapitulate the human condition. In addn., it is difficult to identify crit. cellular and mol. contributors to disease or to vary them independently in whole-animal models. This challenge has attracted the interest of engineers, who have begun to collaborate with biologists to leverage recent advances in tissue engineering and microfabrication to develop novel in vitro models of disease. As these models are synthetic systems, specific mol. factors and individual cell types, including parenchymal cells, vascular cells, and immune cells, can be varied independently while simultaneously measuring system-level responses in real time. In this article, we provide some examples of these efforts, including engineered models of diseases of the heart, lung, intestine, liver, kidney, cartilage, skin and vascular, endocrine, musculoskeletal, and nervous systems, as well as models of infectious diseases and cancer. We also describe how engineered in vitro models can be combined with human inducible pluripotent stem cells to enable new insights into a broad variety of disease mechanisms, as well as provide a test bed for screening new therapies.
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  154. 154
    Li, Z.; Hui, J.; Yang, P.; Mao, H. Microfluidic Organ-on-a-Chip System for Disease Modeling and Drug Development.. Biosensors 2022, 12, 370,  DOI: 10.3390/bios12060370
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    154
    Microfluidic Organ-on-a-Chip System for Disease Modeling and Drug Development
    Li, Zening; Hui, Jianan; Yang, Panhui; Mao, Hongju
    Biosensors (2022), 12 (6), 370CODEN: BIOSHU; ISSN:2079-6374. (MDPI AG)
    An organ-on-a-chip is a device that combines micro-manufg. and tissue engineering to replicate the crit. physiol. environment and functions of the human organs. Therefore, it can be used to predict drug responses and environmental effects on organs. Microfluidic technol. can control micro-scale reagents with high precision. Hence, microfluidics have been widely applied in organ-on-chip systems to mimic specific organ or multiple organs in vivo. These models integrated with various sensors show great potential in simulating the human environment. In this review, we mainly introduce the typical structures and recent research achievements of several organ-on-a-chip platforms. We also discuss innovations in models applied to the fields of pharmacokinetics/pharmacodynamics, nano-medicine, continuous dynamic monitoring in disease modeling, and their further applications in other fields.
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  155. 155
    Park, J.; Wetzel, I.; Marriott, I.; Dréau, D.; D’Avanzo, C.; Kim, D. Y.; Tanzi, R. E.; Cho, H. A 3D Human Triculture System Modeling Neurodegeneration and Neuroinflammation in Alzheimer’s Disease. Nat. Neurosci. 2018, 21, 941,  DOI: 10.1038/s41593-018-0175-4
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    155
    Human 3D triculture system modeling neurodegeneration and neuroinflammation in Alzheimer's disease
    Park, Joseph; Wetzel, Isaac; Marriott, Ian; Dreau, Didier; D'Avanzo, Carla; Kim, Doo Yeon; Tanzi, Rudolph E.; Cho, Hansang
    Nature Neuroscience (2018), 21 (7), 941-951CODEN: NANEFN; ISSN:1097-6256. (Nature Research)
    Alzheimer's disease (AD) is characterized by beta-amyloid accumulation, phosphorylated tau formation, hyperactivation of glial cells, and neuronal loss. The mechanisms of AD pathogenesis, however, remain poorly understood, partially due to the lack of relevant models that can comprehensively recapitulate multistage intercellular interactions in human AD brains. Here we present a new three-dimensional (3D) human AD triculture model using neurons, astrocytes, and microglia in a 3D microfluidic platform. Our model provided key representative AD features: beta-amyloid aggregation, phosphorylated tau accumulation, and neuroinflammatory activity. In particular, the model mirrored microglial recruitment, neurotoxic activities such as axonal cleavage, and NO release damaging AD neurons and astrocytes. Our model will serve to facilitate the development of more precise human brain models for basic mechanistic studies in neural-glial interactions and drug discovery.
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  156. 156
    Lee, H. K.; Velazquez Sanchez, C.; Chen, M.; Morin, P. J.; Wells, J. M.; Hanlon, E. B.; Xia, W. Three Dimensional Human Neuro-Spheroid Model of Alzheimer’s Disease Based on Differentiated Induced Pluripotent Stem Cells. PLoS One 2016, 11, e0163072,  DOI: 10.1371/journal.pone.0163072
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    156
    Three dimensional human neuro-spheroid model of Alzheimer's disease based on differentiated induced pluripotent stem cells
    Lee, Han-Kyu; Sanchez, Clara Velazquez; Chen, Mei; Morin, Peter J.; Wells, John M.; Hanlon, Eugene B.; Xia, Weiming
    PLoS One (2016), 11 (9), e0163072/1-e0163072/23CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
    The testing of candidate drugs to slow progression of Alzheimer's disease (AD) requires clin. trials that are lengthy and expensive. Efforts to model the biochem. milieu of the AD brain may be greatly facilitated by combining two cutting edge technologies to generate three-dimensional (3D) human neuro-spheroid from induced pluripotent stem cells (iPSC) derived from AD subjects. We created iPSC from blood cells of five AD patients and differentiated them into 3D human neuronal culture. We characterized neuronal markers of our 3D neurons by immunocytochem. staining to validate the differentiation status. To block the generation of pathol. amyloid β peptides (Aβ), the 3D-differentiated AD neurons were treated with inhibitors targeting β-secretase (BACE1) and γ-secretases. As predicted, both BACE1 and γ-secretase inhibitors dramatically decreased Aβ generation in iPSCderived neural cells derived from all five AD patients, under std. two-dimensional (2D) differentiation conditions. However, BACE1 and γ-secretase inhibitors showed less potency in decreasing Aβ levels in neural cells differentiated under 3D culture conditions. Interestingly, in a single subject AD1, we found that BACE1 inhibitor treatment was not able to significantly reduce Aβ42 levels. To investigate underlying mol. mechanisms, we performed proteomic anal. of 3D AD human neuronal cultures including AD1. Proteomic anal. revealed specific redn. of several proteins that might contribute to a poor inhibition of BACE1 in subject AD1. To our knowledge, this is the first iPSC-differentiated 3D neuro-spheroid model derived from AD patients' blood. Our results demonstrate that our 3D human neuro-spheroid model can be a physiol. relevant and valid model for testing efficacy of AD drug.
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  157. 157
    Park, J.; Lee, B. K.; Jeong, G. S.; Hyun, J. K.; Lee, C. J.; Lee, S. H. Three-Dimensional Brain-on-a-Chip with an Interstitial Level of Flow and Its Application as an in Vitro Model of Alzheimer’s Disease. Lab Chip 2015, 15, 141,  DOI: 10.1039/C4LC00962B
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    157
    Three-dimensional brain-on-a-chip with an interstitial level of flow and its application as an in vitro model of Alzheimer's disease
    Park, JiSoo; Lee, Bo Kyeong; Jeong, Gi Seok; Hyun, Jung Keun; Lee, C. Justin; Lee, Sang-Hoon
    Lab on a Chip (2015), 15 (1), 141-150CODEN: LCAHAM; ISSN:1473-0189. (Royal Society of Chemistry)
    There has been a growing need for in vitro models of neurodegenerative diseases such as Alzheimer's disease that would enable a better understanding of etiol. and faster development of treatment strategies. However, meeting this demand has been held back by the limited ability to mimic the in vivo microenvironment in an in vitro system. Here, we developed a microfluidic chip based on three-dimensional (3D) neurospheroids that more closely mimics the in vivo brain microenvironment by providing a const. flow of fluid that is readily obsd. in the interstitial space of the brain. Uniform neurospheroids, with cell-cell interactions and contacts in all directions, were formed in concave microwell arrays, and a slow interstitial level of flow was maintained using an osmotic micropump system. Using this platform, we investigated the effect of flow on neurospheroid size, neural network, and neural differentiation. Neurospheroids cultured with flow were larger and formed more robust and complex neural networks than those cultured under static conditions, suggesting an effect of the interstitial level of slow and diffusion-dominant flow on continuous nutrient, oxygen, and cytokine transport and removal of metabolic wastes. We also tested the toxic effects of amyloid-β, which is generally considered to be the major contributor in Alzheimer's disease. Amyloid-β treatment via an osmotic micropump significantly reduced the viability of neurospheroids and caused a significantly more destruction of neural networks, compared to the amyloid-β treatment under static conditions. By adding in vivo-like microenvironments, we propose this 3D culture-based microfluidic chip as an in vitro brain model for neurodegenerative disease and high-throughput drug screening.
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  158. 158
    Li, W.; Alazawi, W. Non-Alcoholic Fatty Liver Disease. Clin. Med. J. R. Coll. Physicians London 2020, 20, 509,  DOI: 10.7861/clinmed.2020-0696
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    Lasli, S.; Kim, H. J.; Lee, K. J.; Suurmond, C. A. E.; Goudie, M.; Bandaru, P.; Sun, W.; Zhang, S.; Zhang, N.; Ahadian, S.; Dokmeci, M. R.; Lee, J.; Khademhosseini, A. A Human Liver-on-a-Chip Platform for Modeling Nonalcoholic Fatty Liver Disease. Adv. Biosyst. 2019, 3, 1900104,  DOI: 10.1002/adbi.201900104
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    Wang, F.; So, K. F.; Xiao, J.; Wang, H. Organ-Organ Communication: The Liver’s Perspective. Theranostics. 2021, 11, 3317,  DOI: 10.7150/thno.55795
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    Organ-organ communication: the liver's perspective
    Wang, Fei; So, Kwok-Fai; Xiao, Jia; Wang, Hua
    Theranostics (2021), 11 (7), 3317-3330CODEN: THERDS; ISSN:1838-7640. (Ivyspring International Publisher)
    A review. Communication between organs participates in most physiol. and pathol. events. Owing to the importance of precise coordination among the liver and virtually all organs in the body for the maintenance of homeostasis, many hepatic disorders originate from impaired organ-organ communication, resulting in concomitant pathol. phenotypes of distant organs. Hepatokines are proteins that are predominantly secreted from the liver, and many hepatokines and several signaling proteins have been linked to diseases of other organs, such as the heart, muscle, bone, and eyes. Although liver-centered interorgan communication has been proposed in both basic and clin. studies, to date, the regulatory mechanisms of hepatokine prodn., secretion, and reciprocation with signaling factors from other organs are obscure. Whether other hormones and cytokines are involved in such communication also warrants investigation. Herein, we summarize the current knowledge of organ-organ communication phenotypes in a variety of diseases and the possible involvement of hepatokines and/or other important signaling factors. This provides novel insight into the underlying roles and mechanisms of liver-originated signal transduction and, more importantly, the understanding of disease in an integrative view.
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  161. 161
    Bauer, S.; Wennberg Huldt, C.; Kanebratt, K. P.; Durieux, I.; Gunne, D.; Andersson, S.; Ewart, L.; Haynes, W. G.; Maschmeyer, I.; Winter, A.; Ämmälä, C.; Marx, U.; Andersson, T. B. Functional Coupling of Human Pancreatic Islets and Liver Spheroids On-a-Chip: Towards a Novel Human Ex Vivo Type 2 Diabetes Model. Sci. Rep. 2017,  DOI: 10.1038/s41598-017-14815-w
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    Herschkowitz, J. I.; Behbod, F. Human Ductal Carcinoma In Situ: From the Eyes of a Beholder. Journal of Mammary Gland Biology and Neoplasia. 2018, 23, 189,  DOI: 10.1007/s10911-018-9419-x
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    Human Ductal Carcinoma In Situ: from the Eyes of a Beholder
    Herschkowitz Jason I; Behbod Fariba
    Journal of mammary gland biology and neoplasia (2018), 23 (4), 189-190 ISSN:.
    There is no expanded citation for this reference.
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    Choi, Y.; Hyun, E.; Seo, J.; Blundell, C.; Kim, H. C.; Lee, E.; Lee, S. H.; Moon, A.; Moon, W. K.; Huh, D. A Microengineered Pathophysiological Model of Early-Stage Breast Cancer. Lab Chip 2015, 15, 3350,  DOI: 10.1039/C5LC00514K
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    163
    A microengineered pathophysiological model of early-stage breast cancer
    Choi, Yoonseok; Hyun, Eunjeh; Seo, Jeongyun; Blundell, Cassidy; Kim, Hee Chan; Lee, Eunhee; Lee, Su Hyun; Moon, Aree; Moon, Woo Kyung; Huh, Dongeun
    Lab on a Chip (2015), 15 (16), 3350-3357CODEN: LCAHAM; ISSN:1473-0189. (Royal Society of Chemistry)
    A mounting body of evidence in cancer research suggests that the local microenvironment of tumor cells has a profound influence on cancer progression and metastasis. In vitro studies on the tumor microenvironment and its pharmacol. modulation, however, are often hampered by the tech. challenges assocd. with creating physiol. cell culture environments that integrate cancer cells with the key components of their native niche such as neighboring cells and extracellular matrix (ECM) to mimic complex microarchitecture of cancerous tissue. Using early-stage breast cancer as a model disease, here we describe a biomimetic microengineering strategy to reconstitute three-dimensional (3D) structural organization and microenvironment of breast tumors in human cell-based in vitro models. Specifically, we developed a microsystem that enabled co-culture of breast tumor spheroids with human mammary ductal epithelial cells and mammary fibroblasts in a compartmentalized 3D microfluidic device to replicate microarchitecture of breast ductal carcinoma in situ (DCIS). We also explored the potential of this breast cancer-on-a-chip system as a drug screening platform by evaluating the efficacy and toxicity of an anticancer drug (paclitaxel). Our microengineered disease model represents the first crit. step towards recapitulating pathophysiol. complexity of breast cancer, and may serve as an enabling tool to systematically examine the contribution of the breast cancer microenvironment to the progression of DCIS to an invasive form of the disease.
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    Ahn, S. I.; Sei, Y. J.; Park, H. J.; Kim, J.; Ryu, Y.; Choi, J. J.; Sung, H. J.; MacDonald, T. J.; Levey, A. I.; Kim, Y. T. Microengineered Human Blood-Brain Barrier Platform for Understanding Nanoparticle Transport Mechanisms. Nat. Commun. 2020,  DOI: 10.1038/s41467-019-13896-7
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    Nair, A. L.; Mesch, L.; Schulz, I.; Becker, H.; Raible, J.; Kiessling, H.; Werner, S.; Rothbauer, U.; Schmees, C.; Busche, M.; Trennheuser, S.; Fricker, G.; Stelzle, M. Parallelizable Microfluidic Platform to Model and Assess in Vitro Cellular Barriers: Technology and Application to Study the Interaction of 3D Tumor Spheroids with Cellular Barriers. Biosensors 2021, 11, 314,  DOI: 10.3390/bios11090314
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    165
    Parallelizable Microfluidic Platform to Model and Assess In Vitro Cellular Barriers: Technology and Application to Study the Interaction of 3D Tumor Spheroids with Cellular Barriers
    Nair, Arya Lekshmi; Mesch, Lena; Schulz, Ingo; Becker, Holger; Raible, Julia; Kiessling, Heiko; Werner, Simon; Rothbauer, Ulrich; Schmees, Christian; Busche, Marius; Trennheuser, Sebastian; Fricker, Gert; Stelzle, Martin
    Biosensors (2021), 11 (9), 314CODEN: BIOSHU; ISSN:2079-6374. (MDPI AG)
    Endothelial and epithelial cellular barriers play a vital role in the selective transport of solutes and other mols. The properties and function of these barriers are often affected in case of inflammation and disease. Modeling cellular barriers in vitro can greatly facilitate studies of inflammation, disease mechanisms and progression, and in addn., can be exploited for drug screening and discovery. Here, we report on a parallelizable microfluidic platform in a multiwell plate format with ten independent cell culture chambers to support the modeling of cellular barriers co-cultured with 3D tumor spheroids. The microfluidic platform was fabricated by microinjection molding. Electrodes integrated into the chip in combination with a FT-impedance measurement system enabled transepithelial/transendothelial elec. resistance (TEER) measurements to rapidly assess real-time barrier tightness. The fluidic layout supports the tubeless and parallelized operation of up to ten distinct cultures under continuous unidirectional flow/perfusion. The capabilities of the system were demonstrated with a co-culture of 3D tumor spheroids and cellular barriers showing the growth and interaction of HT29 spheroids with a cellular barrier of MDCK cells.
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    Steinmetz, K. L.; Spack, E. G. The Basics of Preclinical Drug Development for Neurodegenerative Disease Indications. BMC Neurol. 2009, 9, S2,  DOI: 10.1186/1471-2377-9-S1-S2
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    Zurina, I. M.; Gorkun, A. A.; Dzhussoeva, E. V.; Kolokoltsova, T. D.; Markov, D. D.; Kosheleva, N. V.; Morozov, S. G.; Saburina, I. N. Human Melanocyte-Derived Spheroids: A Precise Test System for Drug Screening and a Multicellular Unit for Tissue Engineering. Front. Bioeng. Biotechnol. 2020,  DOI: 10.3389/fbioe.2020.00540
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    Zhang, Z.; Chen, L.; Wang, Y.; Zhang, T.; Chen, Y. C.; Yoon, E. Label-Free Estimation of Therapeutic Efficacy on 3D Cancer Spheres Using Convolutional Neural Network Image Analysis. Anal. Chem. 2019, 91 (21), 14093– 14100,  DOI: 10.1021/acs.analchem.9b03896
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    168
    Label-Free Estimation of Therapeutic Efficacy on 3D Cancer Spheres Using Convolutional Neural Network Image Analysis
    Zhang, Zhixiong; Chen, Lili; Wang, Yimin; Zhang, Tiantian; Chen, Yu-Chih; Yoon, Euisik
    Analytical Chemistry (Washington, DC, United States) (2019), 91 (21), 14093-14100CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    Despite recent advances in cancer treatment, developing better therapeutic reagents remains an essential task for oncologists. To accurately characterize drug efficacy, 3D cell culture holds great promise as opposed to conventional 2D mono-layer culture. Due to the advantages of cell manipulation in high-throughput, various microfluidic platforms have been developed for drug screening with 3D models. However, the dissemination of microfluidic technol. is overall slow, and one missing part is fast and low-cost assay readout. In this work, we developed a microfluidic chip forming 1,920 tumor spheres for drug testing, and the platform is supported by automatic image collection and cropping for anal. Using conventional LIVE/DEAD staining as ground truth of sphere viability, we trained a convolutional neural network to est. sphere viability based on its brightfield image. The estd. sphere viability was highly correlated with the ground truth (R-value > 0.84). In this manner, we precisely estd. drug efficacy of three chemotherapy drugs, Doxorubicin, Oxaliplatin, and Irinotecan. We also cross-validated the trained networks of Doxorubicin and Oxaliplatin and found common brightfield morphol. features indicating sphere viability. The discovery suggests the potential to train a generic network using some representative drugs and apply to many different drugs in large-scale screening. The brightfield estn. of sphere viability saves LIVE/DEAD staining reagent cost and fluorescence imaging time. More importantly, the presented method allows viability estn. in a label-free and non-destructive manner. In short, with image processing and machine learning, the presented method provides a fast, low-cost, and label-free method to assess tumor sphere viability for large-scale drug screening in microfluidics.
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    Fetah, K. L.; DiPardo, B. J.; Kongadzem, E. M.; Tomlinson, J. S.; Elzagheid, A.; Elmusrati, M.; Khademhosseini, A.; Ashammakhi, N. Cancer Modeling-on-a-Chip with Future Artificial Intelligence Integration. Small 2019, 15 (50), 1901985,  DOI: 10.1002/smll.201901985
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    169
    Cancer Modeling-on-a-Chip with Future Artificial Intelligence Integration
    Fetah, Kirsten Lee; DiPardo, Benjamin J.; Kongadzem, Eve-Mary; Tomlinson, James S.; Elzagheid, Adam; Elmusrati, Mohammed; Khademhosseini, Ali; Ashammakhi, Nureddin
    Small (2019), 15 (50), 1901985CODEN: SMALBC; ISSN:1613-6810. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A review. Cancer is one of the leading causes of death worldwide, despite the large efforts to improve the understanding of cancer biol. and development of treatments. The attempts to improve cancer treatment are limited by the complexity of the local milieu in which cancer cells exist. The tumor microenvironment (TME) consists of a diverse population of tumor cells and stromal cells with immune constituents, microvasculature, extracellular matrix components, and gradients of oxygen, nutrients, and growth factors. The TME is not recapitulated in traditional models used in cancer study, limiting the translation of preliminary findings to clin. practice. Advances in 3D cell culture, tissue engineering, and microfluidics led to the development of "cancer-on-a-chip" platforms that expand the ability to model the TME in vitro and allow for high-throughput anal. The advances in the development of cancer-on-a-chip platforms, implications for drug development, challenges to leveraging this technol. for improved cancer treatment, and future integration with artificial intelligence for improved predictive drug screening models are discussed.
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    Nashimoto, Y.; Okada, R.; Hanada, S.; Arima, Y.; Nishiyama, K.; Miura, T.; Yokokawa, R. Vascularized Cancer on a Chip: The Effect of Perfusion on Growth and Drug Delivery of Tumor Spheroid. Biomaterials 2020, 229, 119547,  DOI: 10.1016/j.biomaterials.2019.119547
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    170
    Vascularized cancer on a chip: The effect of perfusion on growth and drug delivery of tumor spheroid
    Nashimoto, Yuji; Okada, Ryu; Hanada, Sanshiro; Arima, Yuichiro; Nishiyama, Koichi; Miura, Takashi; Yokokawa, Ryuji
    Biomaterials (2020), 229 (), 119547CODEN: BIMADU; ISSN:0142-9612. (Elsevier Ltd.)
    Tumor vasculature creates a hostile tumor microenvironment (TME) in vivo and nourishes cancers, resulting in cancer progression and drug resistance. To mimic the biochem. and biomech. environments of tumors in vitro, several models integrated with a vascular network have been reported. However, the tumor responses to biochem. and biomech. stimuli were evaluated under static conditions and failed to incorporate the effects of blood flow to tumors. In this study, we present a tumor-on-a-chip platform that enables the evaluation of tumor activities with intraluminal flow in an engineered tumor vascular network. The fibroblasts in the tumor spheroid induced angiogenic sprouts, which constructed a perfusable vascular network in a tumor spheroid. The perfusability of the engineered vascular network was preserved during the culture. Moreover, perfusion for over 24 h significantly increased the proliferation activities of tumor cells and decreased cell death in the spheroid. Drug administration under perfusion condition did not show the dose-dependent effects of anticancer drugs on tumor activities in contrast to the results under static conditions. Our results demonstrate the importance of flow in a vascular network for the evaluation of tumor activities in a drug screening platform.
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    Ran, R.; Wang, H. F.; Hou, F.; Liu, Y.; Hui, Y.; Petrovsky, N.; Zhang, F.; Zhao, C. X. A Microfluidic Tumor-on-a-Chip for Assessing Multifunctional Liposomes’ Tumor Targeting and Anticancer Efficacy. Adv. Healthc. Mater. 2019, 8, 1900015,  DOI: 10.1002/adhm.201900015
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    Akay, M.; Hite, J.; Avci, N. G.; Fan, Y.; Akay, Y.; Lu, G.; Zhu, J. J. Drug Screening of Human GBM Spheroids in Brain Cancer Chip. Sci. Rep. 2018,  DOI: 10.1038/s41598-018-33641-2
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    Tavares, R. S. N.; Phuong-Tao, T.; Maschmeyer, I.; Maria-Engler, S. S.; Schäfer-Korting, M.; Winter, A.; Zoschke, C.; Lauster, R.; Marx, U.; Gaspar, L. R. Toxicity of Topically Applied Drugs beyond Skin Irritation: Static Skin Model vs. Two Organs-on-a-Chip. Int. J. Pharm. 2020, 589, 119788,  DOI: 10.1016/j.ijpharm.2020.119788
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    173
    Toxicity of topically applied drugs beyond skin irritation: Static skin model vs. Two organs-on-a-chip
    Tavares, R. S. N.; Phuong-Tao, T.; Maschmeyer, I.; Maria-Engler, S. S.; Schafer-Korting, M.; Winter, A.; Zoschke, C.; Lauster, R.; Marx, U.; Gaspar, L. R.
    International Journal of Pharmaceutics (Amsterdam, Netherlands) (2020), 589 (), 119788CODEN: IJPHDE; ISSN:0378-5173. (Elsevier B.V.)
    Skin model cultivation under static conditions limits the observation of the toxicity to this single organ. Biol.-inspired microphysiol. systems assocg. skin with a liver in the same circulating medium provide a more comprehensive insight into systemic substance toxicity; however, its advantages or limitations for topical substance toxicity remain unknown. Herein, we performed topical (OECD test guideline no. 439) and systemic administration of terbinafine in reconstructed human skin (RHS) vs. a RHS plus liver model cultured in TissUse' HUMIMIC Chip2. Aiming for a more detailed insight into the cutaneous substance irritancy/toxicity, we assessed more than the MTT cell viability: lactate dehydrogenase (LDH), lactate and glucose levels, as well as inherent gene expressions. Sodium dodecyl sulfate (SDS) was the topical irritant pos. control. We confirmed SDS irritancy in both static RHS and Chip2 culture by the damage in the morphol., redn. in the lactate prodn. and lower glucose consumption. In the static RHS, the SDS-treated tissues also released significantly high LDH (82%; p < 0.05) and significantly lower IL-6 release (p < 0.05), corroborating with the other metabolic levels. In both static RHS and Chip2 conditions, we confirmed absence of irritancy or systemic toxicity by LDH, glucose or lactate levels for topical 1% and 5% terbinafine and systemic 0.1% terbinafine treatment. However, topical 5% terbinafine treatment in the Chip2 upregulated IL-1α in the RHS, unbalanced apoptotic and proliferative cell ratios in the liver and significantly increased its expression of CYP1A2 and 3A4 enzymes (p < 0.05), proving that it has passed the RHS barrier promoting a liver impact. Systemic 0.1% terbinafine treatment in the Chip2 increased RHS expression of EGFR, increased apoptotic cells in the liver, downregulated liver albumin expression and upregulated CYP2C9 significantly (p < 0.05), acting as an effective hepatotoxic terbinafine control. The combination of the RHS and liver model in the Chip2 allowed a more sensitive assessment of skin and hepatic effects caused by chems. able to pass the skin (5% terbinafine and SDS) and after systemic 0.1% terbinafine application. The present study opens up a more complex approach based on the microphysiol. system to assess more than a skin irritation process.
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    Cui, X.; Hartanto, Y.; Zhang, H. Advances in Multicellular Spheroids Formation. Journal of the Royal Society Interface. 2017, 14, 20160877,  DOI: 10.1098/rsif.2016.0877
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    Advances in multicellular spheroids formation
    Cui, X.; Hartanto, Y.; Zhang, H.
    Journal of the Royal Society, Interface (2017), 14 (127), 20160877/1-20160877/15CODEN: JRSICU; ISSN:1742-5662. (Royal Society)
    Three-dimensional multicellular spheroids (MCSs) have a complex architectural structure, dynamic cell-cell/cell-matrix interactions and bio-mimicking in vivo microenvironment. As a fundamental building block for tissue reconstruction, MCSs have emerged as a powerful tool to narrow down the gap between the in vitro and in vivo model. In this review paper, we discussed the structure and biol. of MCSs and detailed fabricating methods. Among these methods, the approach in microfluidics with hydrogel support for MCS formation is promising because it allows essential cell-cell/cell-matrix interactions in a confined space.
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  175. 175
    Brown, M. J.; Bahsoun, S.; Morris, M. A.; Akam, E. C. Determining Conditions for Successful Culture of Multi-Cellular 3D Tumour Spheroids to Investigate the Effect of Mesenchymal Stem Cells on Breast Cancer Cell Invasiveness. Bioengineering 2019, 6, 101,  DOI: 10.3390/bioengineering6040101
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    175
    Determining conditions for successful culture of multi-cellular 3D tumour spheroids to investigate the effect of mesenchymal stem cells on breast cancer cell invasiveness
    Brown, Marie-Juliet; Bahsoun, Soukaina; Morris, Mhairi A.; Akam, Elizabeth C.
    Bioengineering (2019), 6 (4), 101CODEN: BIOEBG; ISSN:2306-5354. (MDPI AG)
    Mesenchymal stem cells have been widely implicated in tumor development and metastases. Moving from the use of two-dimensional (2D) models to three-dimensional (3D) to investigate this relationship is crit. to facilitate more applicable and relevant research on the tumor microenvironment. We investigated the effects of altering glucose concn. and the source of fetal bovine serum (FBS) on the growth of two breast cancer cell lines (T47D and MDA-MB-231) and human bone marrow-derived mesenchymal stem cells (hBM-MSCs) to det. successful conditions to enable their co-culture in 3D tumor spheroid models. Subsequently, these 3D multi-cellular tumor spheroids were used to investigate the effect of hBM-MSCs on breast cancer cell invasiveness. Findings presented herein show that serum source had a statistically significant effect on two thirds of the growth parameters measured across all three cell lines, whereas glucose only had a statistically significant effect on 6%. It was detd. that the optimum growth media compn. for the co-culture of 3D hBM-MSCs and breast cancer cell line spheroids was 1 g/L glucose DMEM supplemented with 10% FBS from source A. Subsequent results demonstrated that co-culture of hBM-MSCs and MDA-MB-231 cells dramatically reduced invasiveness of both cell lines (F(1,4) = 71.465, p = 0.001) when embedded into a matrix comprising of growth-factor reduced base membrane ext. (BME) and collagen.
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    Mironov, V.; Visconti, R. P.; Kasyanov, V.; Forgacs, G.; Drake, C. J.; Markwald, R. R. Organ Printing: Tissue Spheroids as Building Blocks. Biomaterials 2009, 30, 2164,  DOI: 10.1016/j.biomaterials.2008.12.084
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    Organ printing: Tissue spheroids as building blocks
    Mironov, Vladimir; Visconti, Richard P.; Kasyanov, Vladimir; Forgacs, Gabor; Drake, Christopher J.; Markwald, Roger R.
    Biomaterials (2009), 30 (12), 2164-2174CODEN: BIMADU; ISSN:0142-9612. (Elsevier Ltd.)
    A review. Organ printing can be defined as layer-by-layer additive robotic biofabrication of 3-dimensional functional living macrotissues and organ constructs using tissue spheroids as building blocks. The microtissues and tissue spheroids are living materials with certain measurable, evolving and potentially controllable compn., material and biol. properties. Closely placed tissue spheroids undergo tissue fusion - a process that represents a fundamental biol. and biophys. principle of developmental biol.-inspired directed tissue self-assembly. It is possible to engineer small segments of an intraorgan branched vascular tree by solid and lumenized vascular tissue spheroids. Organ printing could dramatically enhance and transform the field of tissue engineering by enabling large-scale industrial robotic biofabrication of living human organ constructs with "built-in" perfusable intraorgan branched vascular tree. Thus, organ printing is a new emerging enabling technol. paradigm which represents a developmental biol.-inspired alternative to classic biodegradable solid scaffold-based approaches in tissue engineering.
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    Białkowska, K.; Komorowski, P.; Bryszewska, M.; Miłowska, K. Spheroids as a Type of Three-Dimensional Cell Cultures─Examples of Methods of Preparation and the Most Important Application. International Journal of Molecular Sciences. 2020, 21, 6225,  DOI: 10.3390/ijms21176225
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    Spheroids as a type of three-dimensional cell cultures-examples of methods of preparation and the most important application
    Bialkowska, Kamila; Komorowski, Piotr; Bryszewska, Maria; Milowska, Katarzyna
    International Journal of Molecular Sciences (2020), 21 (17), 6225CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)
    A review. Cell cultures are very important for testing materials and drugs, and in the examn. of cell biol. and special cell mechanisms. The most popular models of cell culture are two-dimensional (2D) as monolayers, but this does not mimic the natural cell environment. Cells are mostly deprived of cell-cell and cell-extracellular matrix interactions. A much better in vitro model is three-dimensional (3D) culture. Because many cell lines have the ability to self-assemble, one 3D culturing method is to produce spheroids. There are several systems for culturing cells in spheroids, e.g., hanging drop, scaffolds and hydrogels, and these cultures have their applications in drug and nanoparticles testing, and disease modeling. In this paper we would like to present methods of prepn. of spheroids in general and emphasize the most important applications.
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  178. 178
    Nishikawa, T.; Tanaka, Y.; Nishikawa, M.; Ogino, Y.; Kusamori, K.; Mizuno, N.; Mizukami, Y.; Shimizu, K.; Konishi, S.; Takahashi, Y.; Takakura, Y. Optimization of Albumin Secretion and Metabolic Activity of Cytochrome P450 1A1 of Human Hepatoblastoma HepG2 Cells in Multicellular Spheroids by Controlling Spheroid Size. Biol. Pharm. Bull. 2017, 40, 334,  DOI: 10.1248/bpb.b16-00833
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    Optimization of albumin secretion and metabolic activity of cytochrome P450 1A1 of human hepatoblastoma HepG2 cells in multicellular spheroids by controlling spheroid size
    Nishikawa, Tomoko; Tanaka, Yutaro; Nishikawa, Makiya; Ogino, Yuka; Kusamori, Kosuke; Mizuno, Narumi; Mizukami, Yuya; Shimizu, Kazunori; Konishi, Satoshi; Takahashi, Yuki; Takakura, Yoshinobu
    Biological & Pharmaceutical Bulletin (2017), 40 (3), 334-338CODEN: BPBLEO; ISSN:0918-6158. (Pharmaceutical Society of Japan)
    Multicellular spheroids are useful as three-dimensional cell culture systems and for cell-based therapies. Their successful application requires an understanding of the consequences of spheroid size for cellular functions. In the present study, we prepd. multicellular spheroids of different sizes using the human hepatoblastoma HepG2 cells, as hepatocytes are frequently used for in vitro drug screening and cell-based therapy. Precise polydimethylsiloxane-based microwells with widths of 360, 450, 560, and 770 μm were fabricated using a micromolding technique. Incubation of HepG2 cells in cell culture plates contg. the microwells resulted in the formation of HepG2 spheroids with av. diams. of 195, 320, 493, and 548 μm. The cell no. per spheroid pos. correlated with its diam., and the viability of HepG2 cells was 94% or above for all samples. The smallest HepG2 spheroids showed the highest albumin secretion. On the other hand, the metabolic activity of 7-ethoxyresorufin, a fluorometric substrate for CYP1A1, increased with increasing spheroid size. These results indicate that controlling spheroid size is important when prepg. HepG2 spheroids and that the size of HepG2 spheroids greatly influences the cellular function of HepG2 cells in the spheroids.
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    Russo, M.; Cejas, C. M.; Pitingolo, G. Advances in Microfluidic 3D Cell Culture for Preclinical Drug Development. Progress in Molecular Biology and Translational Science 2022, 187, 163,  DOI: 10.1016/bs.pmbts.2021.07.022
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    Advances in microfluidic 3D cell culture for preclinical drug development
    Russo, Maria; Cejas, Cesare M.; Pitingolo, Gabriele
    Progress in Molecular Biology and Translational Science (2022), 187 (Micro/Nanofluidics and Lab-on-Chip-Based Emerging Technologyes for Biomedial and Translational Research Applications, Part B), 163-204CODEN: PNARC5; ISSN:1878-0814. (Elsevier Inc.)
    Drug development is often a very long, costly, and risky process due to the lack of reliability in the preclin. studies. Traditional current preclin. models, mostly based on 2D cell culture and animal testing, are not full representatives of the complex in vivo microenvironments and often fail. In order to reduce the enormous costs, both financial and general well-being, a more predictive preclin. model is needed. In this chapter, we review recent advances in microfluidic 3D cell culture showing how its development has allowed the introduction of in vitro microphysiol. systems, laying the foundation for organ-on-a-chip technol. These findings provide the basis for numerous preclin. drug discovery assays, which raise the possibility of using micro-engineered systems as emerging alternatives to traditional models, based on 2D cell culture and animals.
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    Marimuthu, M.; Rousset, N.; St-Georges-Robillard, A.; Lateef, M. A.; Ferland, M.; Mes-Masson, A. M.; Gervais, T. Multi-Size Spheroid Formation Using Microfluidic Funnels. Lab Chip 2018, 18, 304,  DOI: 10.1039/C7LC00970D
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    Multi-size spheroid formation using microfluidic funnels
    Marimuthu, M.; Rousset, N.; St-Georges-Robillard, A.; Lateef, M. A.; Ferland, M.; Mes-Masson, A.-M.; Gervais, T.
    Lab on a Chip (2018), 18 (2), 304-314CODEN: LCAHAM; ISSN:1473-0189. (Royal Society of Chemistry)
    We present a microfluidic platform for automatic multi-size spheroid formation within const. vol. hanging droplets (HDs) from a single inlet loading of a const. cell concn. The platform introduces three technol. improvements over the existing spheroid formation platforms: 1 cell seeding control is achieved by enrichment of a cell soln. rather than diln.; 2 cell seeding in each HD is fully independent and pre-programmable at the design stage; 3 the fabricated chip operates well using a hydrophobic PDMS surface, ensuring long-term storage possibility for device usage. Pre-programmed cell seeding densities at each HD are achieved using a "microfluidic funnel" layer, which has an array of cone-shaped wells with increasing apex angles acting as a metering unit. The integrated platform is designed to form, treat, stain, and image multi-size spheroids on-chip. Spheroids can be analyzed on-chip or easily transferred to conventional well plates for further processing. Empirically, enrichment factors up to 37× have been demonstrated, resulting in viable spheroids of diams. ranging from 230-420μm and 280-530μm for OV90 and TOV112D cell lines, resp. We envision that microfluidic funnels and single inlet multi-size spheroid (SIMSS) chips will find broad application in 3D biol. assays where size-dependent responses are expected, including chemoresponse assays, photodynamic therapy assays, and other assays involving drug transport characterization in drug discovery.
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    Nath, S.; Devi, G. R. Three-Dimensional Culture Systems in Cancer Research: Focus on Tumor Spheroid Model. Pharmacology and Therapeutics. 2016, 163, 94,  DOI: 10.1016/j.pharmthera.2016.03.013
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    Three-dimensional culture systems in cancer research: Focus on tumor spheroid model
    Nath, Sritama; Devi, Gayathri R.
    Pharmacology & Therapeutics (2016), 163 (), 94-108CODEN: PHTHDT; ISSN:0163-7258. (Elsevier)
    Cancer cells propagated in three-dimensional (3D) culture systems exhibit physiol. relevant cell-cell and cell-matrix interactions, gene expression and signaling pathway profiles, heterogeneity and structural complexity that reflect in vivo tumors. In recent years, development of various 3D models has improved the study of host-tumor interaction and use of high-throughput screening platforms for anti-cancer drug discovery and development. This review attempts to summarize the various 3D culture systems, with an emphasis on the most well characterized and widely applied model - multicellular tumor spheroids. This review also highlights the various techniques to generate tumor spheroids, methods to characterize them, and its applicability in cancer research.
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    Anada, T.; Masuda, T.; Honda, Y.; Fukuda, J.; Arai, F.; Fukuda, T.; Suzuki, O. Three-Dimensional Cell Culture Device Utilizing Thin Membrane Deformation by Decompression. Sensors Actuators, B Chem. 2010, 147, 376,  DOI: 10.1016/j.snb.2010.01.065
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    Three-dimensional cell culture device utilizing thin membrane deformation by decompression
    Anada, Takahisa; Masuda, Taisuke; Honda, Yoshitomo; Fukuda, Junji; Arai, Fumihito; Fukuda, Toshio; Suzuki, Osamu
    Sensors and Actuators, B: Chemical (2010), 147 (1), 376-379CODEN: SABCEB; ISSN:0925-4005. (Elsevier B.V.)
    The authors have developed a novel multicellular aggregate (spheroid) culture device that utilizes thin polydimethylsiloxane membrane deformation by decompression. The device was capable of producing thousands of spheroids rapidly at a time and regulating the diam. of spheroids with narrow size distribution. The spheroids were easily retrieved from the device noninvasively. These characteristics of the device suggest that it has the potential for use in a wide variety of applications, such as drug screening and tissue engineering.
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    Ziółkowska, K.; Stelmachowska, A.; Kwapiszewski, R.; Chudy, M.; Dybko, A.; Brzózka, Z. Long-Term Three-Dimensional Cell Culture and Anticancer Drug Activity Evaluation in a Microfluidic Chip. Biosens. Bioelectron. 2013, 40, 68,  DOI: 10.1016/j.bios.2012.06.017
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    Long-term three-dimensional cell culture and anticancer drug activity evaluation in a microfluidic chip
    Ziolkowska, Karina; Stelmachowska, Agnieszka; Kwapiszewski, Radoslaw; Chudy, Michal; Dybko, Artur; Brzozka, Zbigniew
    Biosensors & Bioelectronics (2013), 40 (1), 68-74CODEN: BBIOE4; ISSN:0956-5663. (Elsevier B.V.)
    In this work, we present a microfluidic array of microwells for long-term tumor spheroid cultivation and anticancer drug activity evaluation. The 3-dimensional microfluidic system was obtained by double casting of poly(dimethylsiloxane). Spheroids of HT-29 human carcinoma cells were cultured in the microsystem for 4 wk. After 2 wk of the culture growth slowdown and stop were obsd. and high cell viability was detd. within next 2 wk. The characteristics of a homeostasis-like state were achieved. A cytostatic drug (5-fluorouracil) was introduced into the microsystem with different frequency (every day or every second day) and different concns. The geometry and construction of the microsystem enables flushing away of unaggregated (including dead) cells while viable spheroids remain inside microwells and decreasing spheroid diam. can be obsd. and measured as an indicator of decreasing cell viability. The results have shown differences in response of spheroids to different concns. of 5-fluorouracil. It was also obsd., that higher frequency of drug dosing resulted in more rapid spheroid diam. decrease. The presented microfluidic system is a soln. for cell-based studies in an in vivo-like microfluidic environment. Moreover, observation of decreasing spheroid dimensions is a low-cost, label-free, and easy-to-conduct mean of a quant. detn. of a 3D cellular model response to a applied drug. It is suitable for long-term observation of spheroid response, in a contrary to other viability assays requiring termination of a culture.
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    Kamatar, A.; Gunay, G.; Acar, H. Natural and Synthetic Biomaterials for Engineering Multicellular Tumor Spheroids. Polymers. 2020, 12, 2506,  DOI: 10.3390/polym12112506
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    Natural and synthetic biomaterials for engineering multicellular tumor spheroids
    Kamatar, Advika; Gunay, Gokhan; Acar, Handan
    Polymers (Basel, Switzerland) (2020), 12 (11), 2506CODEN: POLYCK; ISSN:2073-4360. (MDPI AG)
    A review. The lack of in vitro models that represent the native tumor microenvironment is a significant challenge for cancer research. Two-dimensional (2D) monolayer culture has long been the std. for in vitro cell-based studies. However, differences between 2D culture and the in vivo environment have led to poor translation of cancer research from in vitro to in vivo models, slowing the progress of the field. Recent advances in three-dimensional (3D) culture have improved the ability of in vitro culture to replicate in vivo conditions. Although 3D cultures still cannot achieve the complexity of the in vivo environment, they can still better replicate the cell-cell and cell-matrix interactions of solid tumors. Multicellular tumor spheroids (MCTS) are three-dimensional (3D) clusters of cells with tumor-like features such as oxygen gradients and drug resistance, and represent an important translational tool for cancer research. Accordingly, natural and synthetic polymers, including collagen, hyaluronic acid, Matrigel, polyethylene glycol (PEG), alginate and chitosan, have been used to form and study MCTS for improved clin. translatability. This review evaluates the current state of biomaterial-based MCTS formation, including advantages and disadvantages of the different biomaterials and their recent applications to the field of cancer research, with a focus on the past five years.
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    Caliari, S. R.; Burdick, J. A. A Practical Guide to Hydrogels for Cell Culture. Nature Methods. 2016, 13, 405,  DOI: 10.1038/nmeth.3839
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    A practical guide to hydrogels for cell culture
    Caliari, Steven R.; Burdick, Jason A.
    Nature Methods (2016), 13 (5), 405-414CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
    There is growing appreciation of the role that the extracellular environment plays in regulating cell behavior. Mech., structural, and compositional cues, either alone or in concert, can drastically alter cell function. Biomaterials, and particularly hydrogels, have been developed and implemented to present defined subsets of these cues for investigating countless cellular processes as a means of understanding morphogenesis, aging, and disease. Although most scientists concede that std. cell culture materials (tissue culture plastic and glass) do a poor job of recapitulating native cellular milieus, there is currently a knowledge barrier for many researchers in regard to the application of hydrogels for cell culture. Here, we introduce hydrogels to those who may be unfamiliar with procedures to culture and study cells with these systems, with a particular focus on com. available hydrogels.
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    Ferreira, L. P.; Gaspar, V. M.; Mano, J. F. Decellularized Extracellular Matrix for Bioengineering Physiomimetic 3D in Vitro Tumor Models. Trends in Biotechnology. 2020, 38, 1397,  DOI: 10.1016/j.tibtech.2020.04.006
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    Decellularized Extracellular Matrix for Bioengineering Physiomimetic 3D in Vitro Tumor Models
    Ferreira, Luis P.; Gaspar, Vitor M.; Mano, Joao F.
    Trends in Biotechnology (2020), 38 (12), 1397-1414CODEN: TRBIDM; ISSN:0167-7799. (Elsevier Ltd.)
    A review. Recent advances in the extn. and purifn. of decellularized extracellular matrix (dECM) obtained from healthy or malignant tissues open new avenues for engineering physiomimetic 3D in vitro tumor models, which closely recapitulate key biomol. hallmarks and the dynamic cancer cell-ECM interactions in the tumor microenvironment. We review current and upcoming methodologies for chem. modification of dECM-based biomaterials and advanced bioprocessing into organotypic 3D solid tumor models. A comprehensive review of disruptive advances and shortcomings of exploring dECM-based biomaterials for recapitulating the native tumor-supporting matrix is also provided. We hope to drive the discussion on how 3D dECM testing platforms can be leveraged for generating microphysiol. tumor surrogates that generate more robust and predictive data on therapeutic bioperformance.
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    Catoira, M. C.; Fusaro, L.; Di Francesco, D.; Ramella, M.; Boccafoschi, F. Overview of Natural Hydrogels for Regenerative Medicine Applications. J. Mater. Sci. Mater. Med. 2019,  DOI: 10.1007/s10856-019-6318-7
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    Reddy, M. S. B.; Ponnamma, D.; Choudhary, R.; Sadasivuni, K. K. A Comparative Review of Natural and Synthetic Biopolymer Composite Scaffolds. Polymers. 2021, 13, 1105,  DOI: 10.3390/polym13071105
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    A comparative review of natural and synthetic biopolymer composite scaffolds
    Reddy, M. Sai Bhargava; Ponnamma, Deepalekshmi; Choudhary, Rajan; Sadasivuni, Kishor Kumar
    Polymers (Basel, Switzerland) (2021), 13 (7), 1105CODEN: POLYCK; ISSN:2073-4360. (MDPI AG)
    A review. Tissue engineering (TE) and regenerative medicine integrate information and technol. from various fields to restore/replace tissues and damaged organs for medical treatments. To achieve this, scaffolds act as delivery vectors or as cellular systems for drugs and cells; thereby, cellular material is able to colonize host cells sufficiently to meet up the requirements of regeneration and repair. This process is multi-stage and requires the development of various components to create the desired neo-tissue or organ. In several current TE strategies, biomaterials are essential components. While several polymers are established for their use as biomaterials, careful consideration of the cellular environment and interactions needed is required in selecting a polymer for a given application. Depending on this, scaffold materials can be of natural or synthetic origin, degradable or nondegradable. In this review, an overview of various natural and synthetic polymers and their possible composite scaffolds with their physicochem. properties including biocompatibility, biodegradability, morphol., mech. strength, pore size, and porosity are discussed. The scaffolds fabrication techniques and a few com. available biopolymers are also tabulated.
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    Bhatia, S.; Bhatia, S. Natural Polymers vs Synthetic Polymer. Natural Polymer Drug Delivery Systems 2016, 95,  DOI: 10.1007/978-3-319-41129-3_3
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    Zhang, W.; Du, A.; Liu, S.; Lv, M.; Chen, S. Research Progress in Decellularized Extracellular Matrix-Derived Hydrogels. Regenerative Therapy. 2021, 18, 88,  DOI: 10.1016/j.reth.2021.04.002
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    Research progress in decellularized extracellular matrix-derived hydrogels
    Zhang, Wenhui; Du, Aoling; Liu, Shun; Lv, Mingyue; Chen, Shenghua
    Regenerative Therapy (2021), 18 (), 88-96CODEN: RTEHAN; ISSN:2352-3204. (Elsevier B.V.)
    Decellularized extracellular matrix (dECM) is widely used in regenerative medicine as a scaffold material due to its unique biol. activity and good biocompatibility. Hydrogel is a three-dimensional network structure polymer with high water content and high swelling that can simulate the water environment of human tissues, has good biocompatibility, and can exchange nutrients, oxygen, and waste with cells. At present, hydrogel is the ideal biol. material for tissue engineering. In recent years, rapid development of the hydrogel theory and technol. and progress in the use of dECM to form hydrogels have attracted considerable attention to dECM hydrogels as an innovative method for tissue engineering and regenerative medicine. This article introduces the classification of hydrogels, and focuses on the history and formation of dECM hydrogels, the source of dECM, the application of dECM hydrogels in tissue engineering and the com. application of dECM materials.
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  191. 191
    Garreta, E.; Oria, R.; Tarantino, C.; Pla-Roca, M.; Prado, P.; Fernández-Avilés, F.; Campistol, J. M.; Samitier, J.; Montserrat, N. Tissue Engineering by Decellularization and 3D Bioprinting. Materials Today. 2017, 20, 166,  DOI: 10.1016/j.mattod.2016.12.005
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    191
    Tissue engineering by decellularization and 3D bioprinting
    Garreta, Elena; Oria, Roger; Tarantino, Carolina; Pla-Roca, Mateu; Prado, Patricia; Fernandez-Aviles, Francisco; Campistol, Josep Maria; Samitier, Josep; Montserrat, Nuria
    Materials Today (Oxford, United Kingdom) (2017), 20 (4), 166-178CODEN: MTOUAN; ISSN:1369-7021. (Elsevier Ltd.)
    Discarded human donor organs have been shown to provide decellularized extracellular matrix (dECM) scaffolds suitable for organ engineering. The quest for appropriate cell sources to satisfy the need of multiple cells types in order to fully repopulate human organ-derived dECM scaffolds has opened new venues for the use of human pluripotent stem cells (hPSCs) for recellularization. In addn., three-dimensional (3D) bioprinting techniques are advancing towards the fabrication of biomimetic cell-laden biomaterial constructs. Here, we review recent progress in decellularization/recellularization and 3D bioprinting technologies, aiming to fabricate autologous tissue grafts and organs with an impact in regenerative medicine.
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    Kwon, J. S.; Oh, J. H. Microfluidic Technology for Cell Manipulation. Applied Sciences (Switzerland). 2018, 8, 992,  DOI: 10.3390/app8060992
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    Azizipour, N.; Avazpour, R.; Sawan, M.; Rosenzweig, D. H.; Ajji, A. Uniformity of Spheroids-on-a-Chip by Surface Treatment of PDMS Microfluidic Platforms. Sensors and Diagnostics 2022, 1, 750,  DOI: 10.1039/D2SD00004K
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    193
    Uniformity of spheroids-on-a-chip by surface treatment of PDMS microfluidic platforms
    Azizipour, Neda; Avazpour, Rahi; Sawan, Mohamad; Rosenzweig, Derek H.; Ajji, Abdellah
    Sensors & Diagnostics (2022), 1 (4), 750-764CODEN: SDEIAR; ISSN:2635-0998. (Royal Society of Chemistry)
    Spheroids have emerged as a reliable model in preclin. oncol. research. Uniformity of spheroids is the key parameter in the reproducibility and precision of drug test results. Microfluidic-based biochips have many advantages over other spheroid formation methods, including better control over the size of spheroids. Decreasing the cell adhesion to the surface is one of the most important challenges in microfluidic platforms, which could be controlled by appropriate surface engineering methods. We have studied the effect of surface modification of PMDS microfluidic biochips with two commonly used anti-fouling coating materials, BSA and Pluronic F-68, on the uniformity of spheroids produced on-chip. The optimized PDMS surfaces effectively inhibited cell adhesion into the surfaces and promoted cell self-aggregation to produce homogenous and uniform spheroids on-chip. This work highlights the importance of surface modification on the quality and quantity of spheroid formation on microfluidic-based biochips.
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  194. 194
    Ruppen, J.; Wildhaber, F. D.; Strub, C.; Hall, S. R. R.; Schmid, R. A.; Geiser, T.; Guenat, O. T. Towards Personalized Medicine: Chemosensitivity Assays of Patient Lung Cancer Cell Spheroids in a Perfused Microfluidic Platform. Lab Chip 2015, 15, 3076,  DOI: 10.1039/C5LC00454C
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    194
    Towards personalized medicine: chemosensitivity assays of patient lung cancer cell spheroids in a perfused microfluidic platform
    Ruppen, Janine; Wildhaber, Franziska D.; Strub, Christoph; Hall, Sean R. R.; Schmid, Ralph A.; Geiser, Thomas; Guenat, Olivier T.
    Lab on a Chip (2015), 15 (14), 3076-3085CODEN: LCAHAM; ISSN:1473-0189. (Royal Society of Chemistry)
    Cancer is responsible for millions of deaths worldwide and the variability in disease patterns calls for patient-specific treatment. Therefore, personalized treatment is expected to become a daily routine in prospective clin. tests. In addn. to genetic mutation anal., predictive chemosensitive assays using patient's cells will be carried out as a decision making tool. However, prior to their widespread application in clinics, several challenges linked to the establishment of such assays need to be addressed. To best predict the drug response in a patient, the cellular environment needs to resemble that of the tumor. Furthermore, the formation of homogeneous replicates from a scarce amt. of patient's cells is essential to compare the responses under various conditions (compd. and concn.). Here, we present a microfluidic device for homogeneous spheroid formation in eight replicates in a perfused microenvironment. Spheroid replicates from either a cell line or primary cells from adenocarcinoma patients were successfully created. To further mimic the tumor microenvironment, spheroid co-culture of primary lung cancer epithelial cells and primary pericytes were tested. A higher chemoresistance in primary co-culture spheroids compared to primary monoculture spheroids was found when both were constantly perfused with cisplatin. This result is thought to be due to the barrier created by the pericytes around the tumor spheroids. Thus, this device can be used for addnl. chemosensitivity assays (e.g. sequential treatment) of patient material to further approach the personalized oncol. field.
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  195. 195
    Lee, G.; Kim, H.; Park, J. Y.; Kim, G.; Han, J.; Chung, S.; Yang, J. H.; Jeon, J. S.; Woo, D. H.; Han, C.; Kim, S. K.; Park, H. J.; Kim, J. H. Generation of Uniform Liver Spheroids from Human Pluripotent Stem Cells for Imaging-Based Drug Toxicity Analysis. Biomaterials 2021, 269, 120529,  DOI: 10.1016/j.biomaterials.2020.120529
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    195
    Generation of uniform liver spheroids from human pluripotent stem cells for imaging-based drug toxicity analysis
    Lee, Gyunggyu; Kim, Hyemin; Park, Ji Young; Kim, Gyeongmin; Han, Jiyou; Chung, Seok; Yang, Ji Hun; Jeon, Jang Su; Woo, Dong-Hun; Han, Choongseong; Kim, Sang Kyum; Park, Han-Jin; Kim, Jong-Hoon
    Biomaterials (2021), 269 (), 120529CODEN: BIMADU; ISSN:0142-9612. (Elsevier Ltd.)
    Recent advances in pluripotent stem cell technol. provide an alternative source of human hepatocytes to overcome the limitations of current toxicity tests. However, this approach requires optimization and standardization before it can be used as a fast and reliable toxicity screening system. Here, we designed and tested microwell culture platforms with various diams. We found that large quantities of uniformly-sized hepatocyte-like cell (HLC) spheroids (3D-uniHLC-Ss) could be efficiently and reproducibly generated in a short period time from a small no. of differentiating human pluripotent stem cells (hPSCs). The hPSC-3D-uniHLC-Ss that were produced in 500-μm diam. microwells consistently exhibited high expressions of hepatic marker genes and had no significant signs of cell death. Importantly, a hepatic master gene hepatocyte nuclear factor 4α (HNF4α) was maintained at high levels, and the epithelial-mesenchymal transition was significantly attenuated in hPSC-3D-uniHLC-Ss. Addnl., when compared with 3D-HLC-Ss that were produced in other 3D platforms, hPSC-3D-uniHLC-Ss showed significantly higher hepatic gene expressions and drug-metabolizing activity of the enzyme, CYP3A4. Imaging-based drug toxicity studies demonstrated that hPSC-3D-uniHLC-Ss exhibited enhanced sensitivity to various hepatotoxicants, compared to HLCs, which were differentiated under 2D conditions. Precise prediction of drug-induced hepatotoxicity is a crucial step in the early phases of drug discovery. Thus, the hPSC-3D-uniHLC-Ss produced using our microwell platform could be used as an imaging-based toxicity screening system to predict drug hepatotoxicity.
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  196. 196
    Wu, L. Y.; Di Carlo, D.; Lee, L. P. Microfluidic Self-Assembly of Tumor Spheroids for Anticancer Drug Discovery. Biomed. Microdevices 2008, 10, 197,  DOI: 10.1007/s10544-007-9125-8
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    196
    Microfluidic self-assembly of tumor spheroids for anticancer drug discovery
    Wu, Liz Y.; Carlo, Dino; Lee, Luke P.
    Biomedical Microdevices (2008), 10 (2), 197-202CODEN: BMICFC; ISSN:1387-2176. (Springer)
    Creating multicellular tumor spheroids is crit. for characterizing anticancer treatments since it may provide a better model than monolayer culture of in vivo tumors. Moreover, continuous dynamic perfusion allows the establishment of physiol. relevant drug profiles to exposed spheroids. Here we present a physiol. inspired design allowing microfluidic self-assembly of spheroids, formation of uniform spheroid arrays, and characterizations of spheroid dynamics all in one platform. Our microfluidic device is based on hydrodynamic trapping of cancer cells in controlled geometries and the formation of spheroids is enhanced by maintaining compact groups of the trapped cells due to continuous perfusion. It was found that spheroid formation speed (av. of 7 h) and size uniformity increased with increased flow rate (up to 10 μl min-1). A large amt. of tumor spheroids (7,500 spheroids per square centimeter) with a narrow size distribution (10 ± 1 cells per spheroid) can be formed in the device to provide a good platform for anticancer drug assays.
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  197. 197
    Hsiung, L. C.; Chiang, C. L.; Wang, C. H.; Huang, Y. H.; Kuo, C. Te.; Cheng, J. Y.; Lin, C. H.; Wu, V.; Chou, H. Y.; Jong, D. S.; Lee, H.; Wo, A. M. Dielectrophoresis-Based Cellular Microarray Chip for Anticancer Drug Screening in Perfusion Microenvironments. Lab Chip 2011, 11, 2333,  DOI: 10.1039/c1lc20147f
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    197
    Dielectrophoresis-based cellular microarray chip for anticancer drug screening in perfusion microenvironments
    Hsiung, Lo-Chang; Chiang, Chi-Ling; Wang, Chen-Ho; Huang, Yu-Hsu; Kuo, Ching-Te; Cheng, Ji-Yen; Lin, Ching-Hung; Wu, Victoria; Chou, Hsien-Yeh; Jong, De-Shien; Lee, Hsinyu; Wo, Andrew M.
    Lab on a Chip (2011), 11 (14), 2333-2342CODEN: LCAHAM; ISSN:1473-0197. (Royal Society of Chemistry)
    The authors present a dielectrophoresis (DEP)-based cellular microarray chip for cell-based anticancer drug screening in perfusion microenvironments. Human breast cancer cells, MCF7, were seeded into the chip and patterned via DEP forces onto the planar interdigitated ring electrode (PIRE) arrays. Roughly, only one third of the cell amt. was required for the chip compared to that for a 96-well plate control. Drug concns. (cisplatin or docetaxel) were stably generated by functional integration of a concn. gradient generator (CGG) and an anti-crosstalk valve (ACV) to treat cells for 24 h. Cell viability was quantified using a dual staining method. Results of cell patterning show substantial uniformity of patterned cells (92 ± 5 cells per PIRE). Furthermore, after 24 h drug perfusion, no statistical significance in dose-responses between the chip and the 96-well plate controls was found. The IC50 value from the chip also concurred with the values from the literature. Moreover, the perfusion culture exhibited reproducibility of drug responses of cells on different PIREs in the same chamber. The chip would enable applications where cells are of limited supply, and supplement microfluidic perfusion cultures for clin. practices.
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  198. 198
    Elitas, M.; Dhar, N.; Schneider, K.; Valero, A.; Braschler, T.; McKinney, J. D.; Renaud, P. Dielectrophoresis as a Single Cell Characterization Method for Bacteria. Biomed. Phys. Eng. Express 2017, 3, 015005,  DOI: 10.1088/2057-1976/3/1/015005
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    Caglayan, Z.; Demircan Yalcın, Y.; Kulah, H. A Prominent Cell Manipulation Technique in Biomems: Dielectrophoresis. Micromachines 2020, 11, 990,  DOI: 10.3390/mi11110990
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    Giduthuri, A. T.; Theodossiou, S. K.; Schiele, N. R.; Srivastava, S. K. Dielectrophoresis as a Tool for Electrophysiological Characterization of Stem Cells. Biophys. Rev. 2020, 1, 011304,  DOI: 10.1063/5.0025056
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    200
    Dielectrophoresis as a tool for electrophysiological characterization of stem cells
    Giduthuri, Anthony T.; Theodossiou, Sophia K.; Schiele, Nathan R.; Srivastava, Soumya K.
    Biophysics Reviews (Melville, NY, United States) (2020), 1 (1), 011304CODEN: BRIEIM; ISSN:2688-4089. (American Institute of Physics)
    A review. Dielectrophoresis (DEP), a nonlinear electrokinetic technique caused by Maxwell-Wagner interfacial polarization of neutral particles in an electrolyte soln., is a powerful cell manipulation method used widely for various applications such as enrichment, trapping, and sorting of heterogeneous cell populations. While conventional cell characterization and sorting methods require tagging or labeling of cells, DEP has the potential to manipulate cells in a label-free way. Due to its unique ability to characterize and sort cells without the need of labeling, there is renewed interest in using DEP for stem cell research and regenerative medicine. Stem cells have the potential to differentiate into various lineages, but achieving homogeneous cell phenotypes from an initially heterogeneous cell population is a challenge. Using DEP to efficiently and affordably identify, sort, and enrich either undifferentiated or differentiated stem cell populations in a label-free way would advance their potential uses for applications in tissue engineering and regenerative medicine. This review summarizes recent, significant research findings regarding the electrophysiol. characterization of stem cells, with a focus on cellular dielec. properties, i.e., permittivity and cond., and on studies that have obtained these measurements using techniques that preserve cell viability, such as crossover frequency. Potential applications for DEP in regenerative medicine are also discussed. Overall, DEP is a promising technique and, when used to characterize, sort, and enrich stem cells, will advance stem cell-based regenerative therapies. (c) 2020 American Institute of Physics.
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  201. 201
    Delikoyun, K.; Yaman, S.; Yilmaz, E.; Sarigil, O.; Anil-Inevi, M.; Telli, K.; Yalcin-Ozuysal, O.; Ozcivici, E.; Tekin, H. C. HologLev: A Hybrid Magnetic Levitation Platform Integrated with Lensless Holographic Microscopy for Density-Based Cell Analysis. ACS Sensors 2021, 6, 2191,  DOI: 10.1021/acssensors.0c02587
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    201
    HologLev: A Hybrid Magnetic Levitation Platform Integrated with Lensless Holographic Microscopy for Density-Based Cell Analysis
    Delikoyun, Kerem; Yaman, Sena; Yilmaz, Esra; Sarigil, Oyku; Anil-Inevi, Muge; Telli, Kubra; Yalcin-Ozuysal, Ozden; Ozcivici, Engin; Tekin, H. Cumhur
    ACS Sensors (2021), 6 (6), 2191-2201CODEN: ASCEFJ; ISSN:2379-3694. (American Chemical Society)
    In clin. practice, a variety of diagnostic applications require the identification of target cells. D. has been used as a phys. marker to distinguish cell populations since metabolic activities could alter the cell densities. Magnetic levitation offers great promise for sepg. cells at the single cell level within heterogeneous populations with respect to cell densities. Traditional magnetic levitation platforms need bulky and precise optical microscopes to visualize levitated cells. Moreover, the